s2fieldsofstudy: Biology Chemistry

text:
The pharmacological bases of the antiangiogenic activity of paclitaxel

In the mid 1990s, researchers began to investigate the antiangiogenic activity of paclitaxel as a possible additional mechanism contributing to its antineoplastic activity in vivo. In the last decade, a number of studies showed that paclitaxel has antiangiogenic activity that could be ascribed to the inhibition of either tubule formation or cell migration, and to an antiproliferative effect towards activated endothelial cells. Furthermore, paclitaxel was shown to downregulate VEGF and Ang-1 expression in tumor cells, and to increase the secretion of TSP-1 in the tumor microenvironment. Moreover, the new pharmaceutical formulations of paclitaxel (such as liposome-encapsulated paclitaxel, ABI-007, and paclitaxel entrapped in emulsifying wax nanoparticles) enhanced the in vivo antiangiogenic activity of the drug. Thus, the preclinical data of paclitaxel may be exploited to implement a novel and rational therapeutic strategy to control tumor progression in patients.

Introduction
Paclitaxel is a cancer chemotherapeutic agent that is commonly used as first line therapy for many common malignancies, including lung, breast, ovarian and head and neck cancer. It also has high antitumor activity against some uncommon malignancies, such as angiosarcoma and Kaposi sarcoma. Paclitaxel is a member of the taxane class of drugs, and it was the first taxane to enter clinical trials and to receive FDA approval; it is a natural product obtained from the North American Pacific yew tree (Taxus brevifolia). Paclitaxel binds to the beta-subunit of polymerized tubulin and inhibits the dissociation rate of the tubulin subunits from the tubule. Thus, micromolar paclitaxel administration to cells results in the formation of microtubule bundles and asters, arresting cells in mitosis [1].
Besides these known pharmacodynamic properties, medical researchers have discovered a new and unexpected additional characteristic of paclitaxel, one that started a second life for this compound and that created new therapeutic approaches using this molecule: that characteristic is its antiangiogenic activity. Although the anti-vascular effects of tubulin-binding agents have been previously reviewed [2][3][4], the present article focuses solely on the pharmacological bases of the antiangiogenic activity of paclitaxel and provides an extensive and updated overview of the field. Table 1 summarizes the published data on the antiangiogenic effects of paclitaxel in different in vitro and in vivo experimental models.
The first ever published evidence about a possible antiangiogenic activity of paclitaxel was a small report some 15 years ago. Dordunoo and colleagues prepared poly(epsilon-caprolactone) (PCL) microspheres containing paclitaxel, and showed this formulation to inhibit angiogenesis in the chick chorioallantoic membrane (CAM) model [5]. A year later, Belotti et al. [6] demonstrated a strong antiangiogenic activity of paclitaxel, suggesting that this property might contribute to its antineoplastic activity in vivo. The aim of that study was to investigate the effect of paclitaxel on endothelial cell functions, and on angiogenesis. In vivo, paclitaxel (20-28 mg/kg i.v.) significantly Table 1 Pharmacological effects of paclitaxel on angiogenesis

Models
Pharmacological effects at cellular level Pharmacological effects at molecular level References In vitro HUVEC Strong anti-proliferative activity [8] HUVEC Inhibition of cell proliferation, motility and invasiveness in a concentration-dependent manner [6] HMVEC-d, HUVEC Selective inhibition of cell proliferation and induction of apoptosis at low concentrations for prolonged periods of time [18] Human leukemia cell lines VEGF downregulation in vitro (even in drug resistant cells) [9] HUVEC Reduction of the capillary network formation in matrigel [20] Rat fat pad endothelial cells Inhibition of migration [21] HUVEC, rat aortic ring explants Inhibition of cell proliferation, migration, and tube formation at one-tenth the concentration needed to achieve a similar effect on tumor cell lines [22] HUVEC, rat aortic ring explants Inhibition of proliferation, differentiation (tube assay) and induction of cell death [23] HUVEC, HMEC-1 Initiation, without completion, of the mitochondrial apoptotic pathway leading to a slowing down of the cell cycle Cytotoxic effects mediated by microtubule network disturbance, G2-M arrest, increase in Bax/Bcl-2 ratio, and mitochondria permeabilization [40] HUVEC, HMEC-1 Increase of interphase microtubule dynamics in vitro [41] HUVEC, HMVEC-L, HMVEC-D

Inhibition of FGF-2-and VEGF-induced cell proliferation and tube formation in matrigel
Increase of the drug cellular uptake in endothelial cells vs. fibroblasts and tumor cells (mechanism unknown) [35] HUVEC Induction of gene and protein expression of TSP-1 at metronomic concentrations [45] Human ovarian cancer cell lines Decrease of survival factors such as Ang-1 and VEGF [47] Rat bone marrow (BM)derived endothelial progenitor cells (EPC) cell line (TR-BME) Inhibition of tube formation and migration of cells at low concentrations [34] HUVEC Inhibition of migration Increase of the levels of acetylated tubulin; increase of forkhead box O3a translocation into the nucleus [44] In vivo Nude mice bearing murine breast cancer VEGF downregulation [9] Cornea assay Inhibition of FGF-2 and VEGF-induced neovascularization [7] Transgenic murine Met-1 breast cancer model Reduced intratumor angiogenesis VEGF downregulation [9] Human oral squamous cell carcinoma, lung tumor Inhibition of tumor angiogenesis Reduced the immunohistochemical expression of CD31, VEGF and VEGF mRNA [11,12] Melanoma spontaneous metastases model

Inhibition of angiogenesis in melanoma tissue lesions
Reduction of VEGF-A expression [25] Rat Walker 256 breast carcinosarcoma cell xenografts A low dose inhibits bone marrow-derived endothelial progenitor cells (EPC) accumulation at the tumor site and decrease the microvessel density [34] inhibited the angiogenic process in a pellet of matrigel containing tumor cell supernatant, that was injected into mice. In vitro, paclitaxel inhibited endothelial cell proliferation, motility, and invasiveness in a concentrationdependent manner [6]. Interestingly, the authors stated that the antiangiogenic activity of paclitaxel was not linked to its cytotoxicity, since inhibition of endothelial cell chemotaxis and invasiveness occurred at drug concentrations which did not affect endothelial cell proliferation [6]. At the same time, a lower dose of paclitaxel (6 mg/kg i.p.) inhibited the bFGF and VEGF-induced neovascularization of the cornea in mice by 45 and 37 %, respectively [7]. Moreover, Iwahana et al. [8] demonstrated a different chemosensitivity of human umbilical vein endothelial cells (HUVEC) and of tumor-derived endothelial cells from rat KMT-17 fibrosarcoma (TEC) when exposed to the same paclitaxel concentrations. Indeed, paclitaxel had strong anti-proliferative activity against HUVECs, but only weakly inhibited the proliferation of TECs, which expressed greater amounts of P-glycoprotein (P-gp), suggesting a drug resistant phenotype of newly formed capillaries [8].
The first evidence of the antiangiogenic activity of paclitaxel via down-regulation of vascular endothelial growth factor (VEGF) in tumors was obtained in a highly-vascularized transgenic murine Met-1 breast cancer model [9]. Paclitaxel was administered intraperitoneally, at noncytotoxic doses of 0-6 mg/kg/day, to nude mice bearing Met-1 breast tumor. Interestingly, the intratumoral angiogenesis, measured by microvessel tortuosity and microvessel density, was significantly reduced by paclitaxel treatment in a dose-dependent manner. Moreover, paclitaxel also suppressed expression of VEGF in Met-1 tumors transplanted in nude mice [9]. Interestingly, paclitaxel monochemotherapy (175 mg/m 2 i.v. every 21 days for three cycles) significantly decreased VEGF plasma levels in metastatic breast cancer patients with partial response or stable disease, whereas no decline was observed in patients with progressive disease [10]. Paclitaxel also showed an inhibitory effect on tumor angiogenesis in a lung tumor xenograft [11], and in transplanted human oral squamous cell carcinoma [12] models, reducing the immunohistochemical expression of CD31 (an endothelial marker), VEGF, and VEGF mRNA.
However, no unanimous consensus was expressed on the antiangiogenic activity of paclitaxel in vivo. A paclitaxel dosage of 6.25 mg/kg s.c., given five times/week, slowed tumor growth in a CWR22R androgen-independent xenograft model of prostate cancer, without any antiangiogenic effects [13]. Similarly, vascular density was not altered by paclitaxel treatment in an orthotopic pancreatic tumor model [14].

The metronomic concept and paclitaxel
Kerbel et al. [15], at the beginning of this century, coined the term ''accidental angiogenesis inhibitors'' to describe a number of drugs that were tested in clinical trials as possible angiogenesis inhibitors, although they were not originally developed to target tumor angiogenesis. This list of ''accidental'' angiogenesis inhibitors included also established agents such as conventional cytotoxic chemotherapeutic drugs. The antivascular side-effects of chemotherapy were found to be most evident by administering such drugs  [5] Matrigel pellet in mice Inhibition of neovascularization [6] Chick chorioallantoic membrane Inhibition of neovascularization at low concentrations [26] Rat mesentery assay A low dose shortened the length of sprouts in VEGF-mediated angiogenesis [27,28] 4T1 metastatic breast cancer Strong antiangiogenic and antilymphangiogenic activities of low doses [30] Rats bearing syngeneic prostate cancer (Dunning AT-1) not expressing TSP-1 Re-induction of TSP-1 expression in tumors [46] HT-29 colon cancer model; 4T1 metastatic breast cancer Upregulation of TSP-1 expression [29,30] Ovarian carcinoma xenograft model Downregulation of VEGF-B, -D and -A; upregulation of Tie-1, Tie-2 and VEGFR-2 [49] Angiogenesis (2013) 16:481-492 483 continuously, or on a frequent (e.g. weekly or even daily) basis, at concentrations well below their maximum tolerated dose (MTD). This continuous low-dose schedule was defined as metronomic chemotherapy [16]. Such a frequent administration in vitro and in vivo of low doses of chemotherapeutic drugs affects the tumor endothelium and inhibits tumor angiogenesis, with minimal concomitant side effects [17][18][19]. In particular, low concentrations of paclitaxel showed only weak effects on in vitro activated endothelial cells using a short-term exposure (24 h) protocol, whereas a striking toxicity toward vascular endothelial cells was observed by a long-term exposure protocol (144 h) that used very low paclitaxel concentrations [18]. Moreover, a potent differential effect was found at the same paclitaxel concentrations if fibroblasts, and drug-sensitive or multidrug-resistant breast cancer cell lines were used. Indeed, a growth inhibitory effect, as well as induction of apoptosis, was observed with IC 50 values in the range of 25-143 pM for paclitaxel only in the endothelial cells, suggesting a selective antiangiogenic therapeutic window for this taxane [18]. At the same time, Hayot et al. [20] demonstrated that paclitaxel, vincristine and vindesine, at non-cytotoxic concentrations, similarly reduced the capillary network formation by HUVEC cells cultured on Matrigel. Furthermore, paclitaxel was also tested in a chemokinetic migration assay using rat fat pad endothelial cells (RFPECs), showing IC 50 values that were approximately 10 -9 M [21]. The different sensitivity of activated endothelial cells to paclitaxel was also confirmed by other studies. Dicker et al. [22] found that paclitaxel inhibited human endothelial cell proliferation, migration, and tube formation (differentiation) at one-tenth of the concentration needed to achieve a similar effect on tumor cell lines ( Fig. 1). Moreover, similar results were obtained on the proliferation, migration, and differentiation, of cultured human umbilical vein endothelial cells and in the capillary sprouting of rat aortic ring explants, demonstrating that endothelial cells are 10-100-fold more sensitive to paclitaxel than are tumor cells [22]. Additionally, it was demonstrated that angiogenesis could be blocked by paclitaxel, and that this effect was primarily due to inhibition of proliferation and differentiation (as measured by Matrigel assay) and by the induction of cell death [23]. The selectivity of paclitaxel inhibition of cell proliferation is also species specific. Indeed, the inhibition of proliferation of human endothelial cells by paclitaxel was observed at ultra low concentrations (0.1-100 pM), whereas 10 4 to 10 5 -fold higher concentrations were necessary to impact human nonendothelial cells, and nonetheless mouse endothelial cells were insensitive to such ultra low (pM) concentrations of paclitaxel [24]. The antiangiogenic activity of paclitaxel at lower doses was also demonstrated in in vivo models of neovascularization, such as the chick embryo chorioallantoic membrane (CAM) [25], where paclitaxel administered at low concentrations (4, 8, and 12 nM) produced a significant dosedependent antiangiogenic activity [26]. Likewise, using the rat mesentery assay, the effect of paclitaxel at a low, atoxic dose, significantly shortened the length of sprouts at the edge of the expanding network in VEGF165-mediated angiogenesis [27]. Moreover, metronomic paclitaxel significantly suppressed VEGF-A-mediated angiogenesis in the rat mesentery to an extent that closely mirrored the significant increase in prostate AT1 tumor necrosis, and the concomitant decrease in tumor growth rate [28]. Paclitaxel metronomic chemotherapy, in colon cancer xenografts, showed stronger antiangiogenic activity than did paclitaxel administered at the maximum tolerable dose (MTD). In contrast, the MTD chemotherapy induced more apoptotic cells than did the metronomic dosage [29]. More interestingly, Jiang et al. [30] investigated the use of low-dose metronomic (LDM) paclitaxel, as a single agent, in a highly metastatic mouse model of 4T1 breast cancers, and compared it with the maximum tolerable dose (MTD) therapy. LDM therapy displayed a stronger anti-tumor activity in suppressing primary and metastatic breast tumors, with less side effects and stronger anti-angiogenic and anti-lymphangiogenic activities, than MTD therapy. On the other hand, MTD therapy showed stronger proapoptotic and anti-proliferative activities in situ. Based on these results, the authors suggested the use of metronomic paclitaxel therapy to treat advanced breast cancer [30]. Metronomic paclitaxel has been successfully applied in preclinical models of metastatic tumors. In one protocol paclitaxel was injected i.p. at 5 mg/kg per day for 3 weeks to treat preclinical spontaneous melanoma metastasis. This protocol resulted in a relative decrease in spontaneous metastases to the lungs, inhibiting angiogenesis in melanoma tissue lesions and reducing the expression of VEGF-A [31]. Moreover, the weekly paclitaxel administration was also effective in inhibiting angiogenesis, tumor growth, and bone marrow hematopoiesis in a lung cancer model [32]. Indeed, the tumor growth rate was reduced by 50 % and the intratumoral microvasculature was inhibited by 70 %, with a concomitant low degree of leukopenia [32]. This important inhibition of tumor neovascularization by paclitaxel was also confirmed in a model of human breast cancer specimens grown in a fibrin clot. Interestingly, paclitaxel significantly reduced the angiogenic sprouting and the formation of new microvessels if compared to vehicle-treated controls [33]. Furthermore, low dose (non-cytotoxic) paclitaxel inhibited bone marrowderived endothelial progenitor cells accumulation at the tumor site in tumor-bearing rats, and caused a decrease in microvessel density [34] (Fig. 1). A possible explanation for preclinical reports describing the endothelial cell's in vitro sensitivity to paclitaxel, the in vivo antiangiogenic effects of the drug, and clinical reports of the relative efficacy of low weekly doses of paclitaxel in patients refractory to standard (every 21 days) doses of this agent came from the study by Merchan et al. [35]. The authors observed that human endothelial cells can accumulate paclitaxel at more than 5 times greater levels than do normal human fibroblasts and several human cancer cell lines. However, the mechanism by which the endothelial cells accumulate paclitaxel remains to be elucidated. Two possibilities have been suggested: (1) the increased transport of the drug into the cells or, (2) the decrease in the efflux of the drug [35].
The effects of paclitaxel on the inflammatory process (that is a characteristic of cancer progression) and in the tumor immune escape process are still quite controversial. The induction of pro-inflammatory genes and proteins by paclitaxel, especially through the lipopolysaccharide (LPS) signaling pathway, have been reviewed by Fitzpatrick and Wheeler [36]. Moreover, Olsen [37] have described that taxanes (including paclitaxel) promote transcription of the cyclooxygenase (COX)-2 gene and the stabilization of the COX-2 messenger RNA. This pharmacodynamic property provided the rationale for the use of COX-2 inhibitors in combination with taxanes, and preclinical studies have shown enhanced anticancer activity from the addition of COX-2 inhibitors to taxane treatment [37]. However, more recent studies suggest that paclitaxel could modulate tissue factor (TF), as is expressed by tumour-associated endothelial and inflammatory cells (as well as by cancer cells themselves), in human mononuclear cells, in HUVECs, and in the metastatic breast carcinoma cell line MDA-MB-231 [38]. Indeed, paclitaxel significantly reduced the strong TF activity expressed by MDA-MB-231, and the TF levels in mononuclear cells and HUVECs even after the induction by LPS, TNF-a and IL-1b. Interestingly, Napoleone et al. [38] also tested whether paclitaxel could influence IL-6 and IL-1b release from the MDA-MB-231, HUVEC and mononuclear cells, since paclitaxel was shown to induce the expression of inflammatory genes in monocytes and in tumour cells. The authors demonstrated that neither the constitutive expression of IL-6 and IL-1b in MDA-MB-231, nor the basal and LPS-induced release from mononuclear cells or from HUVECs, was affected by paclitaxel administration. Recently, another important finding from Chen et al. [39] underlines the importance of the effects of paclitaxel on immunity. These authors showed that the antitumor activity of metronomic low doses of paclitaxel depends on both its capacity to deplete regulatory T cells, and on its inhibition of tumor angiogenesis. Moreover, the combination of metronomic paclitaxel plus an antigen-specific DNA vaccine induced a more potent antigen-specific immune responses and antitumor effects compared to the vaccine alone [39]. In conclusion, the effect of paclitaxel on inflammation and immunity seems to be more complex than previously hypothesized and surely merits further investigations for its relevant role in the antitumor effect of this taxane.

Molecular mechanisms of paclitaxel antiangiogenic activity
The main molecular mechanisms for the antiangiogenic activity of paclitaxel are graphically summarized in Fig. 2 of paclitaxel on human endothelial cell proliferation: a cytostatic effect at low paclitaxel concentrations, and a cytotoxic effect at higher concentrations [40]. The cytotoxic effect involved primarily those signaling networks that are reported to be impaired in tumor cells (i.e., microtubule network disturbance, G 2 -M arrest, increase in the Bax/Bcl-2 ratio, and mitochondria permeabilization) that result in apoptosis. Conversely, the cytostatic effect of paclitaxel involves the inhibition of endothelial cell proliferation without the induction apoptosis and without any structural modification to the microtubule network. In addition, low paclitaxel concentrations just initiate the apoptotic signaling pathway that is stopped upstream of mitochondria permeabilization and it does not lead to endothelial cell death [40]. Moreover, paclitaxel inhibits angiogenesis by an increase in microtubule dynamics in endothelial cells and by the impairment of interphase microtubule functions [41]. Other studies have uncovered additional mechanisms involved in the antiangiogenic effects of taxanes, including the degradation of heat shock protein 90 [42] and the inhibition of Rac1 and of Cdc42 activity [43]. It should be noted that these data have been obtained with docetaxel, but similar or analogous effects have yet to be reported with paclitaxel. Although the two drugs are very similar and belong to the same antineoplastic class of compounds, no data are available to show that such mechanisms could be responsible for the antiangiogenic activity of paclitaxel.
Recently, Bonezzi et al. [44] found that paclitaxel increases the levels of acetylated tubulin in HUVECs. The induction of tubulin hyperacetylation was concomitant with the inhibition of cell motility but not of cell proliferation, which only occurred at concentrations much higher than those that inhibited cell motility. In fact, the overexpression of a tubulin deacetylase such as the NADdependent histone deacetylase sirtuin-2 (SIRT2), increases cell motility and reduces cell response to the anti-motility activity of paclitaxel [44]. In the same article, Bonezzi et al. [44] found another possible target to explain the antimotility activity of paclitaxel: forkhead box O3a (FOXO3a), a member of the family of the forkhead transcription factor FOXO, that negatively regulate cell motility. Indeed, the authors found that paclitaxel caused the translocation to the nucleus of the FOXO3a, which is a pre-requisite for its transcriptional activity. In 2003 we [45] reported that protracted exposure of endothelial cells in vitro (144 h) to low concentrations of several different anticancer agents, including paclitaxel, caused overexpression of thrombospondin-1 (TSP-1), a potent and endothelial-specific inhibitor of angiogenesis. Elevated circulating TSP-1 levels were also detected in the plasma of mice treated with metronomic chemotherapy and, above all, the antiangiogenic and antitumor effects of low-dose continuous cyclophosphamide were absent in TSP-1-null C57BL/6 mice. These results suggested that TSP-1 acts as a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy [45]. These data were later confirmed in other in vivo models. Indeed, systemic low-dose continuous treatment of a rat malignant prostate cancer model (Dunning AT-1) with paclitaxel induced the expression of TSP-1 in the tumor tissue, and inhibited tumor growth [46]. Moreover, metronomic paclitaxel chemotherapy also caused a dramatic increase in the expression of TSP-1 (whereas MTD chemotherapy did not) in a HT-29 colon cancer model [29] and in the highly metastatic 4T1 mouse model of breast cancer [30]. These findings support the hypothesis that the anti-tumour effect of low-dose metronomic paclitaxel is in part mediated by the induction of TSP-1 (Fig. 2).
Other interesting possible mechanisms include the downregulation of survival factors for endothelial cells such as Angiopoietin-1 (Ang-1) and VEGF-A. Ang-1 and angiopoietin-2 (Ang-2) are major ligands for the endothelium-specific tyrosine kinase receptor Tie-2, and they are important regulators of endothelial cell survival [46]. Long-term exposure (168 h) to low concentrations (2 nM) of paclitaxel significantly decreased Ang-1 and VEGF-A gene and protein expression in human ovarian cancer cell lines [47]. The downregulation of VEGF-A after paclitaxel treatment was also observed in tissue samples from 39 patients (diagnosed with invasive carcinoma) that were cultured with paclitaxel for 24 h. The mean VEGF-A levels were significantly lower in the treated samples compared to controls [48]. Moreover, the downregulation of VEGF-B and -D was also reported in response to paclitaxel treatment in ovarian carcinoma xenografts, whereas the expressions of Tie-1 and Tie-2 were significantly upregulated. In the same study, paclitaxel treatment significantly decreased the expression of VEGF-A, and it increased the expression of VEGFR-2 [49] (Fig. 2).

New pharmaceutical formulations of paclitaxel enhance its antiangiogenic activity
In the last decade numerous efforts have focused on removing the use of Cremophor EL to decrease the hypersensitive side effects, or to improve the pharmacokinetic behavior, of paclitaxel. There are a variety of nanoparticle carrier systems being explored for chemotherapeutic drugs, including paclitaxel, such as liposomes, pegylated liposomes, protein nanoparticles, and polymeric nanoparticles [50].
Liposomes represent an established drug delivery system for lipophilic substances, and they act by improving the pharmacokinetics and the therapeutic index of the anticancer drugs [51]. Indeed, liposomal paclitaxel showed improved solubility and similar in vitro cytotoxicity against a variety of tumor cell lines compared to that of the Cremophor EL paclitaxel preparation [52]. However, a major limit in the clinical use of conventional liposome is its rapid clearance by the reticuloendothelial system after systemic administration, and is low bioavailability [53].
In contrast to the conventional liposomes, positively charged cationic liposomes have been developed to be preferentially bound to, and internalized by angiogenic and negatively charged, endothelial cells that are found in tumours and in areas of chronic inflammation [54]. Indeed, Thurston et al. [54] have shown that actively growing angiogenic endothelial cells exhibit a preferential uptake of cationic liposomes by endocytosis.
In 2003, Schmitt-Sody et al. [55] evaluated a novel neovascular targeting therapy, consisting of paclitaxel encapsulated in cationic liposomes, on tumor selectivity and antitumoral efficacy in comparison with the paclitaxel free drug in vivo ( Table 2). The endothelial deposition of paclitaxel in tumor vessels significantly increased during the infusion time (compared to Cremophor EL paclitaxel preparation) and both the subcutaneous tumor growth and the appearance of regional lymph node metastases were significantly delayed in a amelanotic hamster melanoma model [55]. Around the same time, also Kunstfeld et al. [56] demonstrated that paclitaxel encapsulated in cationic liposomes, which have been shown to target blood vessels, prevented melanoma growth and invasive behavior, which overall resulted in an increase in the survival of the tumorbearing mice. Moreover, liposome-encapsulated paclitaxel reduced vessel density at the interface between the tumor and the dermis, and reduced endothelial cell mitosis. In contrast, equimolar concentrations of paclitaxel solubilized in Cremophor EL produced only weak effects on tumor growth and did not reduce the mitotic index of the endothelium in vivo [56]. These data were also confirmed in an animal model of prostate cancer. Thus, paclitaxel encapsulated in cationic liposomes (EndoTAG-1) caused a significant decrease of microvessel density in tumors when compared to paclitaxel alone, confirming the relative suppression of angiogenesis when compared to the conventional treatment [57] (Table 2).
Another promising approach to the synthesis of liposomes with prolonged half-life is the grafting of the liposome with the inert and biocompatible polymer polyethylene glycol (PEG). This modification prevents the recognition of liposome by opsonins and therefore reduces its clearance by cells of the reticuloendothelial system [58]. The pegylated liposome is therefore often referred to as a sterically stabilized liposome (SSL). Huang et al. [59] evaluated the antiangiogenic activity of sterically stabilized liposomes containing paclitaxel (SSL-PTX). SSL-PTX effectively inhibited endothelial cell proliferation and migration in a concentration-dependent manner and its metronomic administration induced marked tumor growth inhibition in the MDA-MB-231 xenograft model via an antiangiogenic mechanism (i.e., reduced microvessel density), when compared to the injection of paclitaxel formulated in Cremophor EL [59].
In order to improve the activity of low concentrations of Cremophor EL-free paclitaxel on endothelial cell proliferation, motility, and tube formation, and to facilitate the delivery to blood vessels, new nanoparticles have been synthesized and tested in vitro and in vivo ( Table 2). Colloidal carriers have been shown to improve tumor therapy by increased drug delivery to tumors resulting directly from the enhanced permeability and retention of the drug. Thus, paclitaxel entrapped in emulsifying wax nanoparticles (PTX NPs) showed enhanced efficacy against colon cancer xenograft models, overcoming paclitaxel resistance and increasing the drug's antiangiogenic effect [60]. Moreover, the development of polymeric nanospheres (NPs), that are able to selectively target the activated vascular endothelium and to deliver co-encapsulated anti-angiogenic agents, has also been studied with paclitaxel [61]. Indeed, NPs loaded with paclitaxel have been successfully tested for their antiangiogenic efficacy in HUVECs in vitro, and in rat aorta rings ex vivo. These effects were more potent than those observed with free paclitaxel [61].
Albumin is a very attractive drug carrier for antineoplastic drugs [62]. It has been demonstrated that albumin helps endothelial transcytosis of protein-bound and unbound plasma constituents, mainly via a cell-surface glycoprotein receptor (gp60). The gp60 protein binds to caveolin-1 a remarkable retardation of tumor growth and appearance of regional lymph node metastases [55] Paclitaxel encapsulated in cationic liposomes In vivo metastatic melanoma model Inhibition of newly blood vessels, prevention of melanoma growth and invasiveness, improvement of mice survival [56] Paclitaxel encapsulated in cationic liposomes (EndoTAG-1)

In vivo prostate tumor xenograft
Decrease of microvessel density [57] Sterically stabilized liposomes containing paclitaxel (SSL-PTX) HUVEC proliferation and migration in vitro; MDA-MB-231 breast cancer xenograft models Inhibition of cell proliferation and migration at low concentrations; decrease of microvascular density of tumors treated with low doses of paclitaxel [59] Paclitaxel entrapped in emulsifying wax nanoparticles (PTX NPs) Colon cancer xenograft models Increase of antiangiogenic effect in the colon cancer xenograft models [60] Polymeric nanospheres loaded with paclitaxel In vitro HUVEC culture and ex vivo rat aortic rings Inhibition of proliferation and inhibition of endothelial sprouts [61] ABI-007, a cremophor EL-free, albumin-bound, 130-nm form of paclitaxel Rat aortic rings, human endothelial cell proliferation and tube formation.

Tumor xenografts
Inhibition of rat aortic microvessel outgrowth, human endothelial cell proliferation, and tube formation.

MCF-7 tumor xenografts Antitumor and antiangiogenic effects in vivo.
Decrease of microvessel density [68] (an intracellular protein) which results in the subsequent formation of transcytotic vesicles. In addition, gp60 also binds to osteonectin. Both caveolin-1 and osteonectin are frequently present in certain neoplasms (such as breast, lung, and prostate cancer), which could account for the accumulation of albumin in some tumors. Such accumulation of albumin could facilitate intratumoral accumulation of albumin-bound drugs [62]. In that respect, albuminbound paclitaxel ABI-007 is a novel, cremophor EL-free, 130-nm particle formulation of paclitaxel [63]. This formulation is obtained by high-pressure homogenization of paclitaxel with serum albumin, resulting in a nanoparticle colloidal suspension [64]. Preclinical studies on human breast cancer xenografts have demonstrated that ABI-007 has a higher penetration into tumor cells, with increased antitumor activity, compared to an equal dose of standard paclitaxel [63]. Interestingly, the antiangiogenic activity of ABI-007 was described in the pivotal study by Ng et al. [65], who employed a therapeutic metronomic approach. Indeed, recent data showed that clinically relevant concentrations of vehicles such as Cremophor EL and polysorbate 80 nullify the antiangiogenic activity of taxanes [66]. These results could hamper the usefulness of paclitaxel metronomic regimens. In order to solve this problem, Ng et al. [65] tested ABI-007 both in in vitro and in in vivo models (Table 2). ABI-007 was found to significantly inhibit rat aortic microvessel outgrowth, human endothelial cell proliferation, and tube formation. Moreover, metronomic ABI-007 significantly suppressed tumor growth in xenograft models with a very low toxicity profile [65].
Recently, hyaluronic acid (HA) conjugates of paclitaxel (HA-PTX) were shown to produce strong anti-tumor effects when dosed metronomically. They also induced anti-angiogenic effects of greater magnitude than those achieved with MTD administration, or with free PTX administration in female nude mice bearing ovarian cancer cells [67]. Moreover, in a taxane-resistant model of ovarian cancer, a significant reduction in tumor weight was noted in the metronomic HA-PTX treated groups, whereas the response of the MTD group was not statistically significant. Metronomic HA-PTX treatment resulted in marked increases in TSP-1, confirming the anti-angiogenic mechanism of action of this novel formulation [67].
Another interesting new paclitaxel formulation was published by Liang et al. [68]. The novel paclitaxel conjugate (PEG-VC-PABC-PTX) was designed and synthesized using p-aminobenzylcarbonyl (PABC), a spacer, and valine-citrulline (VC), a substrate of cathepsin B, to link polyethylene glycol (PEG) and PTX. The PEG-VC-PABC-PTX showed significant antitumor and anti-angiogenic effects in vivo, while the control conjugate was almost ineffective [68].
The development of an oral formulation of paclitaxel is necessary for the clinical development of metronomic chemotherapy. Moes et al. [69] have increased the oral bioavailability of paclitaxel by combining ritonavir with a new oral solid dispersion formulation of paclitaxel (1/11 w/w paclitaxel, 9/11 w/w polyvinylpyrrolidone K30, and 1/11 w/w sodium lauryl sulfate)-which they termed ModraPac001. Currently, the ModraPac001 formulation is being applied in the first clinical trial with oral metronomic chemotherapy of paclitaxel [69].
Among all the new formulations of paclitaxel with demonstrated antiangiogenic characteristics, ABI-007 was the most successful in clinical practice. Indeed, it was initially approved by the US Food and drug Administration in January 2005, and indicated for use with pretreated metastatic breast cancer patients. ABI-007 is currently approved in 42 countries, including the European Union, Canada, India and Japan [64]. Thus, in a relatively short period, there has been an impressive improvement in our understanding of the antiangiogenic mechanism of paclitaxel. Further laboratory and clinical research is necessary to maximize the benefits of the antiangiogenic properties of paclitaxel for the treatment of patients with various cancers. In this context, the knowledge and investigation of metronomic chemotherapy based on the new formulations of paclitaxel, such as ABI-007, HA-PTX or ModraPac001, constitutes today one of the most exciting strategies for improving the clinical control of angiogenesis in primary and metastatic tumors [70]. On the other hand, such treatment strategies should be based on solid and significant results of randomized phase III clinical trials, and not only on their safety profiles or on their impact on the quality of life and on patient preference. Nonetheless, tolerability and compliance will probably become the most important factors in the future, according to the emerging overall importance of quality of life in cancer care [71]. The availability of new drugs for metronomic chemotherapeutic strategies (i.e. ABI-007, HA-PTX and ModraPac001), alone or in association with other targeted molecules, will give the oncologist many different effective treatment options for metastatic patients that are already resistant to standard doses of chemotherapy [70]. Indeed, both preclinical [72] and clinical studies [73,74] have suggested that metronomic chemotherapy could be effective in stabilizing tumors already resistant to the same drug MTD treatments. Moreover, the ability of paclitaxel to inhibit different pathways of the angiogenic process may be a great advantage in the treatment of tumors already resistant to antiangiogenic drugs with a specific target (e.g. bevacizumab or sunitinib).

Conclusion
The aim of this review was to summarize the published data on the molecular and pharmacological bases of the antiangiogenic activity of paclitaxel, and to underline this particular pharmacodynamic characteristic of the drug for possible future clinical development. In fact, paclitaxel (and its novel formulations) both at MTD and at low dose schedules demonstrated significant antiangiogenic activity. This effect could be ascribed both to a cytotoxic or to a cytostatic activity towards activated endothelial cells, or to the inhibition of capillary formation and cell migration. Moreover, paclitaxel has been shown to inhibit the release of VEGF and Ang-1 by tumor cells, and to increase the secretion of TSP-1 to the tumor microenvironment. It is therefore possible that the different antiangiogenic mechanisms of paclitaxel described in this review may explain the antiangiogenic activity of this drug in different human tumors, such as prostate and breast cancer. Indeed, the angiogenic process in these tumors may depend on the balance of different proangiogenic factors and endogenous angiogenic inhibitors. Paclitaxel can definitely affect those different pathways, resulting in an overall clinical activity, and one that is independent from the direct cytotoxic effects of the drug on cancer cells. Thus, based on the available published preclinical data, paclitaxel (and especially its novel formulations) may permit novel and rational therapeutic strategies to control tumor progression in patients.


extfieldsofstudy: Biology Medicine

text:
Loss of Function of the Mouse Sharpin Gene Results in Peyer’s Patch Regression

Peyer’s patches (PP) are an important component in the immune response against intestinal pathogens. Two independent, spontaneous mutations in the mouse Sharpin gene (Sharpincpdm and Sharpincpdm-Dem) result in the absence of PP and disrupted splenic white pulp in adult mice, although a full complement of lymph nodes is present. Here we report that rudimentary PP begin to develop in Sharpincpdm mice during embryogenesis, but lack the organizational patterns that are typical of this tissue. In the present study, small intestines examined at weekly intervals from birth to maturity showed spontaneous regression of PP in mutant mice with concurrent infiltration of granulocytes. At 5 to 6 weeks of age, only indistinct remnants of granulocytic accumulations remain. Transplantation of normal bone marrow into Sharpincpdm mice at 7 days of age did not prevent regression of PP in bone marrow chimeras examined at 7 to 8 weeks of age. These findings indicate that SHARPIN expression is required for the normal development and maintenance, but not initiation, of PP.

Introduction
Peyer's patches (PP) are organized secondary lymphoid tissues located in the anti-mesenteric wall of the small intestine of mammals. They comprise the major inductive sites for adaptive immune responses to intestinal antigens. PP are an important source of IgA-producing B cells that develop through class switching from IgM + cells under the influence of T cells in the germinal centers of PP, although T cell-independent IgA production also occurs [1].
PP development (reviewed in [2]) is a nuclear factor kappa B (NFKB)-dependent process initiated in the intestinal stroma of embryonic mice by vascular cell adhesion molecule 1 (VCAM1) + , intracellular adhesion molecule 1 (ICAM1) + stromal organizer cells on the antimesenteric surface of the small intestine at 15.5 days post conception (dpc), followed by recruitment of lymphoid tissue inducer (LTi) cells. LTi cells express multiple surface markers, including CD4, PTPRC (CD45R or B220), IL7R, and CXCR5 (BLR1). Interactions between IL7R signal-induced lymphotoxin (LT) alpha1beta2 expression on LTi cells and the LT beta receptor (LTBR) on organizer cells activate NFKB transcription factors, driving chemokine expression to create the microenvironment for further PP development. Compartmentalization of PP anlagen into biologically efficient structures is initiated near the end of gestation in mice, prior to the entry of lymphocytes. Recruitment of mature B and T cells begins after 18.5 days post conception (dpc), and segregation of B and T cell compartments is completed postpartum [3].
C57BL/KaLawRij-Sharpin cpdm /RijSunJ mutant mice have severe, progressive multiorgan inflammation [4]. PP are absent in adult (examined at 6+ weeks of age) Sharpin cpdm mice, as determined by serial sections of Swiss rolls of the small intestine, and accordingly fecal IgA levels are greatly reduced [5]. The spleen has decreased white pulp, lacks a defined marginal zone, and B cell function is impaired, with significantly reduced serum IgG, IgA, and IgE. Although all lymph nodes are present, the remaining secondary immune organs lack follicular dendritic cells (FDCs), B cell follicles, and germinal centers [5,6].
To begin to elucidate the mechanism underlying the absence of PP in adult Sharpin cpdm mutant mice, the small intestines of neonatal and young mice homozygous for the recessive Sharpin cpdm mutation were examined for evidence of PP anlagen. Surprisingly, PP populated by B and T lymphocytes were present in neonatal Sharpin cpdm mice, although they were smaller in size and lacked the organization seen in PP of mice heterozygous or wildtype for Sharpin (controls). Spontaneous regression of PP in maturing Sharpin cpdm mice was typically associated with infiltration by granulocytes.

Results
Clusters of ICAM1 + cells were observed by whole mount immunohistochemistry (WMI) in the small intestines of neonatal (0-1 day postpartum) mutant and control mice. There was no significant difference in the number of these patches (mean, 4.7 and 5.6 in mutants and controls, respectively), although increased labeling intensity and compartmentalization of these cells could be readily observed in PP of control, but not mutant, mice (Fig. 1A, B).
B220-positive foci were readily identified at the anti-mesenteric surface of the small intestines of neonatal mice by WMI. At 1 to 2 weeks of age, multiple individual follicles could be discerned in the PP of control mice. The size of follicles and of individual PP subjectively increased with age in control mice. Representative findings are shown in Fig. 1C. B220-positive foci were also present in the intestine of mutant mice between 0 days and 5 weeks of age, however, these did not contain apparent follicular structures. They were less organized, smaller, and less prominent than those of the controls, and the intensity of the labeling became variable and overall decreased greatly by 3 weeks of age. Although organized PP could easily be identified and B220 + staining was still present in control PP up to 6 weeks of age this method became less reliable (with less uniform staining) in older mice.
Sections of the small intestine were evaluated by microscopic examination of hematoxylin and eosin (H&E) stained Swiss rolls of the small intestine, confirming the presence of PP in young mutant and control mice. These patches did not have defined follicles in mutant mice, in contrast to controls. In some PP of mutant mice at 2 to 3 weeks of age, there were increased numbers of nonlymphoid cells, including granulocytes, stromal cells, and macrophages. Non-lymphoid cells comprised a small fraction of the cells in PP of the controls. At 4 and 5 weeks of age, a dramatic change was seen in the size and cell population of the few remaining patches in Sharpin cpdm mice, in that they were composed of sparse numbers of lymphocytes admixed with granulocytes, macrophages, and increased amounts of fibrous connective tissue (Fig. 2). In mutant mice aged 6 weeks and older, no distinct PP were seen using this method. However, occasional remnants of granulocytic accumulations were observed in the antimesenteric submucosa of the small intestine, presumably the sites of degenerate PP.
To determine the composition and organization of PP remnants in young mutant mice, frozen sections of the intestine were examined using indirect immunofluorescence (IFA) microscopy with antibodies against CD45RB and CD3 for B and T cells, respectively. Similar to results obtained via other methods, PP of mutant mice appeared smaller than the PP of controls at all time points, and fewer PP were typically found. In addition, mutant PP remnants were consistently less organized, with little compartmentalization of B and T cells and no follicular structures observed. As mice matured, increasing numbers of non-B, non-T cells were observed in the PP remnants of mutant mice, consistent with the infiltration of granulocytic populations observed by H&E staining. By contrast, the PP of control mice subjectively increased in size with age, and exhibited distinct compartments, with CD3 + T cells mostly concentrated in the interfollicular areas and occasionally seen in B cell follicles (Fig. 3).
The splenic white pulp of adult Sharpin cpdm mutant mice is disorganized with poor separation of B and T cell areas [5]. To determine whether the observed changes in the PP of mutant mice parallel changes in the organization of white pulp, frozen sections of the spleen were examined in mutant and control mice at weekly intervals up to 5 weeks of age, identifying cell populations as described for the intestine. While differences were subtle in mice ,2 weeks of age, the white pulp in mutant mice appeared smaller and more dispersed than that of controls at all time points. White pulp size and organization subjectively increased in the spleens of control mice as they matured. At 4 and 5 weeks of age, the white pulp of Sharpin cpdm mutant mice became progressively more disorganized with increased mixing of B and T cell populations and scattering of B cells along with increased numbers of non-lymphoid cells. Splenomegaly caused by increased extramedullary hematopoiesis was common in mutant mice. In control mice, segregation of the red and white pulp was more distinct, and the white pulp contained numerous lymphocytes. Representative images are shown in Fig. 4.
To further evaluate whether loss of PP may be attributable to defects in hematopoietic cells, stromal cells, or both, bone marrow cells from healthy histocompatible donor mice transgenically expressing green fluorescent protein (GFP) + were transferred into irradiated Sharpin cpdm and control mice at 7 days of age. Recipient mice (6 mutants and 8 controls of both sexes) were euthanized and necropsied at 7 to 8 weeks of age. Engraftment of donor cells was confirmed by immunohistochemistry for GFP + cells [7] in the spleen, liver, colon, and cecum. Although the skin phenotype of mutant mice was subjectively milder than expected (ie, mild to moderate rather than moderate to severe) in most recipents, no evidence of reconstitution of PP was detected histologically (by examination of Swiss rolls of the intestine as described) in any of the recipient mutant mice. Normal PPs were found in recipient control mice histologically, and repopulation of lymphoid tissues including the gut associated lymphoid tissues of the large intestine   and cecum by donor cells was subsequently confirmed by immunohistochemistry for GFP + cells.

Discussion
The development of PP during late embryogenesis has been well characterized in recent years. This varies from development of other secondary lymphoid organs in some key ways. In lymph node organogenesis, early lymph sacs bud from vascular endothelium in a prospero-related homeobox 1 (PROX1)-dependent manner to accumulate LTi cells, while PP are initiated by the Ret proto-oncogene-dependent clustering of previously scattered LTi cells with KIT + , ITGAX (CD11C) + initiator cells in the small intestine [8,9]. The local microenvironments for these tissues require different cytokines and signaling pathways to induce lymphoid development. Whereas PP require JAK3-dependent IL7R signaling, lymph nodes require TRAF6-dependent TNFSF11 (TRANCE) signaling for the differentiation and function of LTi cells [10][11][12]. Additionally, the signaling requirements for cervical and mesenteric lymph nodes can vary from those of other lymph nodes (reviewed in [13]). Development of splenic white pulp is not dependent on LTi cells, but its organization and maturation requires activation of NFKB through LTA1B2 signaling in lymphocytes [14]. Similarly, although pathways involving TNF, TNFRSF1A, LTA, LTB, TNFSF14 (LIGHT), and the LTBR all have complex roles in the development and organization of secondary lymphoid organs [15][16][17][18][19][20][21], LTA1B2 activation of NFKB transcription factors via the LTBR is indispensible for lymph node and PP development. The LTBR is one of several tumor necrosis factor (TNF) superfamily receptors, the binding of which lead to activation of inhibitor of kappa B kinases (IKKs) and MAP3K14 (NFKB inducing kinase [NIK]); these phosphorylate inhibitory proteins that sequester NFKB homo-and heterodimers in the cytoplasm, and subsequent ubiquitin-proteasome pathway degradation of the inhibitors allows nuclear translocation of NFKB complexes for transcription of target genes (reviewed in [22]). Among genes downstream of the LTBR and other TNF superfamily receptors are those involved in inflammation, cell survival, and innate immunity (primarily through canonical activation of the NFKB1-P50 heterodimer), lymphoid tissue development (primarily through noncanonical activation of the NFKB2-P52 heterodimer), and organization of these structures (involving both pathways).
SHARPIN was recently discovered to be component of the RBCK1 (HOIL1L) and RNF31 (HOIP)-containing linear ubiquitin chain assembly complex. Linear ubiquitination of the IKK regulatory subunit IKBKG (NEMO) is among the functions of this complex, and SHARPIN deficiency was shown to result in impaired CD40-and TNFA-induced activation of the IKK complex and subsequent canonical NFKB activation in B cells and mouse embryo fibroblasts in vitro [33][34][35]. Although activation of the noncanonical NFKB pathway, which is essential for secondary lymphoid organ development, was not reported to be affected in B cells or mouse embryo fibroblasts, SHARPIN may potentially influence this pathway in other cell types or in specialized microenvironments during lymphoid tissue development. Investigators demonstrated that ablation of TNF in Sharpin cpdm mutant mice rescued the inflammatory skin phenotype and eliminated granulocyte accumulations in various organs, but did not rescue development of the splenic marginal zone or PP [33]. Thus, the inflammatory cell infiltrates observed in regressing PP of young mice in the present study are not likely to be the cause of PP loss. Dysregulation of local chemokine expression in high endothelial venules and stromal tissues may contribute to the accumulation of granulocytes in secondary lymphoid organs in this disease.
Findings of the present study demonstrate that while PP development is initiated in Sharpin cpdm mutant mice, essential compartmentalization which normally begins prior to the entry of lymphocytes fails. Efficient segregation into B and T cells areas is lacking, and the previously described absence of FDCs, possibly a consequence of the faulty B cell maturation or defects in stromal FDC precursors, leads to absence of B cell follicles [36,37]. Changes in the spleen, although less severe, parallel those observed in the PPs. Increased extramedullary myelopoiesis accompanies accumulation of granulocytes in the spleens of mutant mice over time. Whereas organization, compartmentalization, and lymphocyte accumulations increase with maturity in spleens of controls, a more primitive white pulp structure is evident in mutant mice, with increased mixing of B and T cells beginning between 2 and 4 weeks of age, and failure of these compartments to expand.
Cooperative signaling between stromal tissues and hematopoetic cells regulates cell survival, differentiation, and proliferation, as well as the segregation and trafficking of B and T lymphocytes in secondary lymphoid organs during development and in immune responses. Loss of one or more of these signals likely leads to the lack of immunologically competent compartments and eventual regression of PP in Sharpin cpdm mutant mice.
Sharpin is conserved among several species, including mice and humans [38]. Such evolutionary conservation suggests a biological advantage. Results of this and other studies suggest that SHARPIN has essential roles in secondary lymphoid organ development, maturation of B cells and subsequent immunoglobulin production, and control of inflammation. At least some of the developmental abnormalities observed in Sharpin cpdm mutant mice in the present study appear to be caused by the absence of SHARPIN in stromal cells, because the compartmentalization of PP stromal cells is independent of lymphocyte entry [3]. Furthermore, irradiation of juvenile Sharpin cpdm mutant mice and transplantation with bone marrow from healthy donors at a time when PP are present did not prevent complete regression of PP by 8 weeks of age.
Despite a lack of normal organization, lymphocytes were recruited into PP anlagen in neonatal mutant mice, indicating that SHARPIN is essential for the organization and maintenance, but not the initial development of PP.

Mice
All experiments involving mice (breeding, genotyping, bone marrow transplantation, and euthanasia) followed procedures approved by The Jackson Laboratory Animal Care and Use Committee under ACUC approvals 07005 and LDS 02-03.

Evaluation of PP Development in Juvenile Mice
Mice used for IFA, immunohistochemistry, and WMI evaluations were F2 and F3 offspring from a cross between Sharpin cpdm mutant males and wildtype females of a congenic strain (C.CAST-(D15Mit156-D15Mit2)/Sun), created for fine mapping of the Sharpin gene [39,40]. Mice carrying C57BL/KaLawRij-Sharpin cpdm /RijSunJ alleles at markers flanking the Sharpin region were identified as mutants. Mice carrying CAST/EiJ alleles, or heterozygotes, were identified as controls. At least 3 mice per group were examined for each time point. Although no gross effect on the phenotype was observed in the mapping cross, to verify that the phenomenon of PP regression was not attributable to background genetics of the congenic mapping cross, we examined sections of the small intestine of C57BL/KaLawRij-Sharpin cpdm / RijSunJ mice at 4 to 7 weeks of age. Results were similar to those seen in mice from the congenic mapping cross (data not shown).
WMI was performed as previously described with minor modifications [3,41], using antibodies to ICAM1 (biotinylated, Pharmingen, 3E2) or B220 (biotinylated, Pharmingen C363.16A). Briefly, tissues collected at necropsy were immediately fixed in 4% paraformaldehyde on ice. After 30 minutes, tissues were transferred into 4% glycine on ice for 30 minutes, then gradually dehydrated in methanol (MeOH). Blocking was done in 30% H 2 O 2 in MeOH for 30 minutes, followed by blocking in PBS with 2% dry milk powder and 0.1% Triton-X 100 (PBMST) twice for 1 hour at RT. Biotinylated primary antibody was applied at 2 ug/ mL in PBMST overnight at 4C. After 5 washes in PBMST at 4C, antibodies were detected using ABC kit (Vectastain Elite, Vector Labs) at a 1:20 dilution in PBS, for 2 hours at 4C. After 5 washes in PBMST at 4C, and a final wash in PBST at RT, diaminobenzidine solution was applied at 250 mg/mL (with 0.05% CoCl 2 in some assays) until a color change was observed, followed by copious washing in PBST. Tissues were gradually dehydrated in MeOH and examined and photographed using an Olympus SZX12 dissecting microscope and Olympus DP70 digital camera. Counts of ICAM1 + patches were analyzed for significance by Student's t-test and Welch's t-test.
For histologic evaluation of cross-sections, Swiss rolls of small intestine collected at necropsy were prepared using a modification of methods previously described [42]. Paraffin-embedded, Fekete's acid alcohol formalin fixed tissues were sectioned at 5 mm and stained with H&E. For immunofluorescence, a 5-cm segment of small intestine proximal to the cecum in mice .2 weeks of age, or Swiss rolls of the small intestine in younger mice, and spleens were prepared in Optimal Cutting Temperature (OCT, 4583, Sakura, Torrance CA, USA) compound. The small intestine was removed at necropsy into PBS on ice, then infused with a mixture of OCT diluted with 5% sucrose in PBS prior to embedding in OCT and freezing on dry ice. Blocks were stored at 280C until use, sectioned at 8 mm and collected on Superfrost Plus glass slides (Fisher, Rockville, IL, USA). Slides were fixed in acetone at 220C for 10 minutes and air dried at room temperature for 15 minutes before washing in PBS, and then placed in an incubation/ humidity chamber. Tissue was incubated with hamster anti-mouse CD3e (clone 500A2, Pharmingen, NJ, USA) and rat anti-mouse CD45RB (protein tyrosine phosphatase, receptor type, C, isoform B; clone 16A, Pharmingen) with 3% FBS 2 hours at room temperature. Slides were washed in PBS and incubated with secondary antibody (AlexaFluor 488 goat anti-rat IgG, AlexaFluor 546 goat anti-hamster IgG, Molecular Probes, Invitrogen, Carlsbad, CA, USA) in PBS and then counterstained with 49,6-Diamidino-2-phenylindole, dialactate (DAPI; D9564, Sigma, St. Louis, MO, USA). Slides were washed in PBS, cover slipped with Fluoromount mounting medium (F4680, Sigma), and examined and photographed using a Leica DMRXE microscope and DFC300FX camera or Leica SP5 AOBS confocal microscope.
Femurs and tibias of donor C57BL/6-Tg(UBC-GFP)30Scha/J mice were surgically removed with sterile forceps and scissors immediately after euthanasia by CO 2 asphyxiation. Bones were placed in a sterile petri dish containing 5 mL of sterile Hanks Buffered Saline Solution (HBSS) with 10% FBS. A sterile mortar and pestle were used to gently crush the bones, and material was placed in a sterile 50 mL tube and vortexed to release the cells and allow bone fragments settle to the bottom of the tube. The supernatant was decanted and poured through a 70 mm mesh strainer; 10 mL of HBSS was added to the filtered supernatant and the process was repeated once. Cells were centrifuged, the supernatant discarded, and red blood cells were lysed with 10 mL of ammonium chloride lysis buffer (0.15M NH 4 Cl, 10 mM KHCO3, 0.1 mM Na 2 EDTA, pH 7.4) on ice for 10 minutes. The remaining cells were rinsed twice with HBSS (1 to 5 mL) and viable cells were counted with a hemacytometer.
Recipient Sharpin cpdm (n = 8; 4 females and 4 males) and control mice (8; 5 females and 3 males) were irradiated at 500 cGy by use of a Cs irradiator as previously described [44]. Mice were then injected intraperitoneally with 1610 7 prepared bone marrow cells from homozygous GFP + donors. Mice were observed daily for one week following engraftment and twice weekly from then on. By 1.5 weeks of age hair loss was evident and at 3 weeks of age mice were completely hairless. Hair regrowth was detected at 4 weeks of age and appeared complete by 6 weeks of age.
Two male Sharpin cpdm mice died at 8 weeks of age and tissues were not available for necropsy. Following euthanasia at 7 to 8 weeks of age, complete necropsies were performed in the remaining 6 Sharpin cpdm (4 female and 2 male) and 8 control mice (5 female and 3 male). Tissues were collected at necropsy and placed in Fekete's acid alcohol formalin solution. Slides were prepared from paraffin-embedded tissues (5 mm thick sections, including Swiss rolls of the intestine), routinely stained with H&E, and read by a veterinary pathologist (JPS). The pathologist was not informed of identification and genotype of the mice at the time of evaluation; however, skin lesions considered typical of the Sharpin cpdm phenotype were evident to some degree in all mutant mice, thus evaluation could not be performed in a completely blinded manner. Engraftment was confirmed in all recipients by detection of GFP + cells as previously described [7] in the spleen, liver, colon, and cecum.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Gender differences in microcirculation: Observation using the hamster cheek pouch

OBJECTIVES: Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation. METHODS: Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). RESULTS: The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17–22) leaks/cm2 vs. 124 (123–128) leaks/cm2, respectively, p<0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73–102)% vs. 64 (55–69)%, p<0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups. CONCLUSION: The female gender could have a direct protective role in microvascular reactivity and the increase in macromolecular permeability induced by ischemia/reperfusion.

& INTRODUCTION
The incidence of cardiovascular disease differs significantly between men and women during their fertile period. Epidemiological studies have revealed that atherosclerosis, hypertension, and peripheral vascular and coronary artery diseases occur with greater prevalence in men and postmenopausal women compared with women at a fertile age (1,2). The reasons for these differences are subject to debate, but it is conceivable that the absolute levels of estradiol and the different degrees of estrogen receptor regulation are the two major determinant factors. However, genetic differences may also exert effects independently of gonadal function (3).
Estrogen has been shown to play an important protective role in non-reproductive systems such as the cardiovascular system. Estrogen causes vasodilation through non-genomic actions, resulting in rapid increases in nitric oxide (NO) production as well as through genomic actions by inducing transcription of NO synthase. These effects on NO bioavailability provide estrogen with the ability to improve arterial wall responsiveness following vascular injury and to inhibit matrix deposition (4). Estrogen may also decrease systemic vascular resistance, improve coronary and peripheral endothelial function, and prevent coronary artery spasm in women with and without coronary atherosclerosis (5). The endothelium effects on the modulation of vasorelaxation are thought to also occur through an endothelium-derived hyperpolarizing factor by inducing vasodilator prostanoids and by inhibiting endothelin-1 production (6,7). Additionally, endothelium action could modulate myogenic vascular responses, resulting in the reduction of microvessel basal tone (8). The majority of these effects have been attributed to estrogen acting on two ligand-activated transcription factors, ERa and ERb, and one G-protein coupled receptor (GPR30 or GPER) expressed on vascular endothelial and smooth muscle cells (4).
The purpose of this study was to further explore the previous findings detailing the cardiovascular protective effects of endogenous estrogens and to focus on gender specificities in the in vivo microcirculation.

& METHODS
Experiments were performed on 36 male and 36 female hamsters (Mesocricetus auratus), which were divided into six animals/group. Group I was used to evaluate macromolecular permeability, group II was used to evaluate leukocyteendothelium interactions, and the subsequent groups were used to assess microvascular reactivity as follows: group III, phenylephrine (a 1 -agonist with vasoconstrictor properties); group IV, endothelin-1 (potent endothelial-derived vasoconstrictor); group V, acetylcholine (endothelial-dependent vasodilator); and group VI, sodium nitroprusside (endothelial-independent vasodilator). All animals were at a fertile age and were matched for age, without any attempt to classify females according to their menstrual cycle. Experiments were performed using animals at 21 weeks of age, according to the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised in 1996), and the protocol was approved by the Ethical Committee of the State University of Rio de Janeiro. All animals received the same appropriate isocaloric laboratory diet (Nuvital from Nuvilab, Curitiba, PR, Brazil).
On the day of the experiment, anesthesia was induced with 0.1-0.2 ml of sodium pentobarbital (Pentobarbital sodique, Sanofi, Paris, France, 60 mg/ml), which was administered intraperitoneally and maintained with a-chloralose [1,2-O-(2,2,2-trichlorethyliden) a-D-glucofuranose, Merck, Darmstadt, Germany] at a dose of 150 mg/kg body weight/h, administered through a femoral vein catheter. The femoral artery was also cannulated to enable pressure measurements. Throughout and following surgery, the temperature of the animals was kept at 37.5˚C with a heating pad controlled by a rectal thermistor (LTB 750 Thermostat System, Uppsala process data AB, Sweden). A tracheal tube was inserted to facilitate spontaneous breathing.
Conceptually, the hamster cheek pouch has both skeletal muscle and cutaneous microcirculatory beds, and in this study, experiments were performed with the cutaneous tissue to facilitate the comparison with data available in the literature (9,10). This part of the pouch is highly vascularized, and all classes of microcirculatory vessels can usually be visualized in the microscopic field with good clarity, thereby allowing for the comparison of effects in different microvascular segments.

Cheek pouch preparation
Cheek pouch preparations were dissected according to the protocol of Duling (11), as modified by Bouskela and Grampp (12), and mounted in an experimental chamber, where they were continuously superfused at a rate of 4 ml/min by a HEPES-supported HCO 2 3 buffered saline solution (composition in mM: NaCl 110.0, KCl 4.7, CaCl 2 2.0, MgSO 4 1.2, NaHCO 3 18.0, HEPES 15.39, and HEPES Na + -salt 14.61) bubbled with 5% CO 2 /95% N 2 to keep the pH of the superfusate at 7.40. Throughout the experiments, the same gas mixture was also gently blown over the experimental chamber to keep the pO 2 level at 12-15 mmHg, monitored by an O 2 electrode in the superfusion solution, as previously described (12). The temperature of the solution was kept at 36.0¡0.5˚C with a circulating bath (model 8005, Polyscience, Niles, Illinois, USA) and (Leitz optical magnification 4006, Wetzlar, Hessen, Germany) coupled to a closed-circuit TV system and allowed to rest for 30 min. The cheek pouch preparation is stable with respect to microvessel diameter, spontaneous arteriolar vasomotion, arteriolar blood flow, and functional capillary density for at least 4 h of microscopic observation (12).

Ischemia/reperfusion of the cheek pouch
Local ischemia of the cheek pouch was obtained by a cuff made of a thin latex tubing that was mounted around the neck of the everted pouch where it exits the mouth of the hamster; this procedure was previously validated in our laboratory (9,10). The placement of the cuff can be made without any visible interference with local blood flow. The intratubular pressure can be rapidly increased by air compression using a syringe and also rapidly decreased at evacuation. An intratubular pressure of 200-220 mmHg in the cuff resulted in a complete arrest of microvascular blood flow, which eventually returned to a level similar to that observed before occlusion. In these experiments, the duration of the ischemia was 30 min.

Increase in macromolecular permeability after ischemia/reperfusion
To quantify the increase in macromolecular permeability, 30 min after completion of the preparative procedure, fluorescein isothiocyanate (FITC)-dextran, with a substitution of two FITC molecules per 1,000 glucose molecules in the polysaccharide chain, was administered at a dose of 25 mg/ 100 g as an intravenous injection of a 5% solution in 0.9% saline. The sites of leakage were defined as fluorescent spots with a diameter of approximately 40 mm found near postcapillary venules. Extravasated material is continuously washed away by the superfusion solution, and consequently, the fluorescent spots gradually decrease (13). Before the ischemic period, hamsters in which the prepared area showed spontaneous nonfading leaks or more than 10 fading leaks were discarded. The number of leaks was counted 10 min after the ischemic period because it was shown that the maximum number of leaks is observed at this time (9,14).

Leukocytes rolling or sticking after ischemia/ reperfusion
Three venular segments with a diameter of approximately 40 mm each were selected for observation. Leukocytes were labeled in vivo with rhodamine G (10 mg/100 g body mass) immediately before observation, and images were recorded on VHS tapes for later analysis. Rolling was defined as the occurrence of contact between the leukocyte and the venular wall, i.e., when the speed of the leukocyte in the bloodstream was lower than that observed for red blood cells. A leukocyte was considered as adherent when it did not show any visible movement for at least 30 s (15).

Measurements of arteriolar reactivity
For the evaluation of microvascular reactivity, a preparation was considered suitable for experimentation if, under control conditions, there was an indication of good vascular tone (which, in 15 separate control tests, implied that the arteriolar diameter could be increased by 56¡3% through topical application of papaverine, 10 mg/ml), a brisk blood flow in all parts of the vascular bed including the larger veins (where individual erythrocytes should not be discernible in the blood stream images), and no tendency for leukocytes to adhere to the venular wall. Phenylephrine, endothelin-1, acetylcholine, and sodium nitroprusside (Sigma Chemicals, St. Louis, MO, USA) were freshly prepared for each experiment and applied topically, added to the superfusion solution, to the cheek pouch with a syringe infusion pump 22 (model 25-2222, Harvard Apparatus, MA, USA) to avoid systemic effects, such as changes in blood pressure, which could result in biased results. For consecutive measurements, three arterioles were selected in each preparation, taking into account the potential for returning them to exactly the same site (based on, e.g., the presence of fat cells and bifurcations). Experiments were performed by taking 3-min videotape recordings of selected microvessels under initial control conditions (before the addition of any drug) and 10-15 min after each experimental intervention. The cumulative concentrations of phenylephrine and endothelin-1 used were 10 29 , 10 27 , and 10 25 M, in order to create a dose-response curve, while the concentrations of acetylcholine and sodium nitroprusside used were 10 28 , 10 26 , and 10 24 M. Diameter measurements were performed using an Image Shearing Device (model 907, Bela Vista, CA, USA).

Statistical analysis
StatSoft Statistica 8.0 software was used for statistical analysis, and variables were tested regarding the possible problems associated with data distribution (i.e., normality, kurtosis, skewness, and homoscedasticity).
Results are shown as medians and quartile ranges, with the exception of weight and blood pressure, which are shown as the means and standard deviations. Increases in macromolecular permeability, number of adherent or rolling leukocytes, and arteriolar diameters were analyzed by gender using the pairwise non-parametric Mann-Whitney U test, and non-parametric analysis of variance using the Kruskal-Wallis and Dunn's tests for post hoc comparisons was performed for comparisons of different concentrations and times. The significance level (a) used in all tests was 0.05.
Arteriolar diameter values were transformed to percentages relative to the initial diameter measurements taken as baseline values, i.e., before the addition of any drug to each arteriole associated with each preparation.

& RESULTS
Seventy-two animals (21 weeks old), 36 males and 36 females, with a mean body mass and blood pressure, respectively, of 124.7¡24.2 vs. 141.1¡39.5 g (NS) and 95.2¡1.2 vs. 95.0¡0.8 mmHg (NS). All six study groups were compared according to gender, and the only group that presented any difference in baseline parameters was group V (acetylcholine), in which females had a higher body mass than males (187.7¡10.4 vs. 145.2¡7.2 g; p,0.01).
Before the addition of any drug, the baseline arteriolar diameters were similar between the groups. Males and females showed, respectively, the following initial arteriolar diameters: phenylephrine group, 34

& DISCUSSION
Ischemia of the cheek pouch for 30 min followed by reperfusion is able to induce leukocyte adhesion and increase plasma extravasations in post-capillary venules (16). In this study, we observed a significant decrease in microvascular permeability to macromolecules in females compared with males without a concomitant reduction in the number of rolling or adherent leukocytes.
Endothelial cells (ECs) form a barrier between blood and tissue environments and control the movement of blood and immune cells, plasma fluid, small molecules, and proteins between the vascular compartment and the extracellular space. In continuous, non-fenestrated endothelium, the  (17). The paracellular pathway is the most understood route, where junctional complexes are composed of tight (TJ), adherens (AJ), and gap junctions that create a physiological intercellular barrier. It has become increasingly clear that the changes/shifts in the function and regulation of these respective TJ and AJ proteins contribute to paracellular regulation and are important for vascular integrity and function (18,19). Additionally, the role of estrogens in these structures is now under investigation. Mitochondrial oxidative stress caused by reactive oxygen species (ROS) generation after ischemia/reperfusion injury has been shown to regulate the mitochondrial release of cytochrome C into the cytosol, leading to the activation of caspase-3, which cleaves the endothelial cell AJ protein bcatenin (20). The cleavage of b-catenin is thought to be a key regulator of cell-cell adhesion and therefore the loss of this important AJ protein results in barrier dysfunction and hyperpermeability. 17b-estradiol has been shown to inhibit important components of the intrinsic apoptotic signaling pathway, causing increased mitochondrial (ROS) formation, decreased mitochondrial transmembrane potential, mitochondrial release of cytochrome C, and activation of caspase-3 (21). In humans, another mechanism contributing to the antioxidant effect of estrogens was observed in young women recruited for in vitro fertilization, where increased estrogen levels led to enhanced manganese superoxide dismutase (SOD) and extracellular (EC)-SOD expression in circulating monocytes (22). It should also be noted that a previous study demonstrated that the administration of SOD isoforms, specifically EC-SOD and copper-zinc SOD, was able to reduce the post-ischemic permeability increase in the hamster cheek pouch, demonstrating that reperfusion injury could be related to the formation of ROS (23).
Although we have not been able to prove the mechanisms involved in estrogen protection, our results suggest that the decreased microvascular permeability observed in females could be dependent on an up-regulation of transmembrane proteins, leading to reinforcement of the structural integrity of TJs and AJs, as well as inhibition of all important components of the intrinsic apoptotic signaling pathway. However, this hypothesis remains to be validated.
The control of reactivity and tonus strongly depends on endothelial and smooth muscle cells and their interactions. These cells express adrenergic receptors (a, e, b, and their subtypes) in variable amounts, depending on the vascular bed. Sympathetic neurons release catecholamines, and their effects depend on the type of receptor activated; for example, stimulation of a 1 receptors (e.g., by phenylephrine) promotes vasoconstriction (24). Animal and human models have shown that there are gender differences in adrenergic vasomotor responses, although the results are conflicting. Some studies did not show significant differences in vasoreactivity after adrenergic stimuli between genders, while other studies demonstrated that the female vasculature is less responsive to norepinephrineand phenylephrine-induced vasoconstriction (25)(26)(27). It is possible that these conflicting results could be explained by different concentrations of circulating sex hormones.  Sex hormone receptors are present in many vascular beds and act through genomic and non-genomic mechanisms. Among these receptors, it appears that the estrogen receptor has the most prominent action due to its potent endothelialdependent vasodilatation effect and vasoconstriction resistance (28). A comparison of castrated and non-castrated male rats demonstrated that there was no difference in vascular contraction following castration; however, when comparing intact and ovarectomized female rats, the latter demonstrated increased vasoconstriction in response to stimuli (8).
The present study showed that, in both genders, there was significant, dose-dependent vasoconstriction induced by phenylephrine but that this effect was less pronounced in females, thereby supporting previous findings (25,26). Contrary to previous studies (29-31), we did not detect any gender differences with regard to secondary vascular responses to acetylcholine, sodium nitroprusside, and endothelin-1. These findings could be due to the heterogeneity observed in the microcirculation in vivo, where several physiological mechanisms are involved simultaneously and may reveal different results. Additionally, there are controversial studies concerning the action of endothelin-1, with some researchers observing reduced vasoconstriction in males (30) and other researchers observing it in females (32). In our study, the vasoconstriction elicited by endothelin-1 was not significantly different between males and females. With respect to microvascular reactivity after acetylcholine administration, the literature has more consistent positive findings in females (29), which we were unable to reproduce in our study. It is possible that the higher body mass of females compared with males may have biased our results in acetylcholine group. Finally, it is well-known that the action of an endothelium-independent stimulus is less evident when an atherosclerotic process is already present. It is possible that if the animals had been older or had a pro-atherosclerotic disease, such as diabetes mellitus, we may have found gender differences at microcirculation after sodium nitroprusside administration. Other limitations of the study should also be highlighted. The unawareness of the menstrual cycle period of females may have influenced the microvascular reactivity (33), although some researchers still consider this a controversial issue (34). Moreover, the reduced number of animals in each group may have limited our ability to observe differences.
Gender differences in the microvascular bed were observed in this study, and according to our findings, we conclude that the female gender seemed to exhibit microvascular protection, exhibited as lower microvascular permeability to macromolecules induced by ischemia/ reperfusion and reduced microvascular constriction elicited by a phenylephrine stimulus.

& ACKNOWLEDGMENTS
This study was supported by grants from the National Research Council of Brazil (CNPq) and the Agency for Research Support of Rio de Janeiro State (FAPERJ). The authors do not have any conflicts of interest to declare.

& AUTHOR CONTRIBUTIONS
Panazzolo DG analyzed the data and wrote the manuscript. Silva LH wrote the manuscript. Cyrino FZ generated data. Sicuri FL analyzed data. Kraemer-Aguiar LG analyzed the data and wrote the manuscript. Bouskela E analyzed the data, wrote the manuscript and designed the research approach.


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Revealing the Mechanism of In Vitro Wound Healing Properties of Citrus tamurana Extract

In the present investigation, we examined the effect of Hyuganatsu (Citrus tamurana) extract (HE) on skin fibroblast (TIG-119) proliferation and migration during in vitro wound healing. HE selectively inhibited proliferation of TIG-119 cells at higher concentration (>1.0 mg/mL); at lower concentrations (0.1, 0.25, 0.5, and 0.75 mg/mL), it exhibited linear and time-dependent cell proliferation. In vitro scratch wound healing studies showed that the HE also accelerated the migration of cells towards the wounded region. Cytometric analysis demonstrated that HE extract did not alter G1/0 and S phases of cell cycle in any concentration studied; however, G2/M phases of cell cycle were significantly (P < 0.05) accelerated at 0.75 mg/mL dose. RT-PCR and Western blotting analysis indicated that HE markedly overexpressed levels of Rac-1, Rho-A, and Cdc-42 mRNA and the respective proteins. Cyclin-dependent kinases (Cdk-1 and -2) gene expression activity was significantly (P < 0.05) increased, but protein content decreased during treatment with HE. The induction of Cdk-1 and -2 by HE was abolished by inhibitors, transcription (DRB), and translation (CHX), implying transcriptional regulation that required de novo protein synthesis.

Introduction
Cell migration and proliferation coupled with controlled cell cycle are beneficial for the repair of sagged and wrinkled skin, dermal, and gastrointestinal wound healing. Cell cycle is a conserved proliferative signaling cascade pathway in mammals and comprises the G 1 , S, G 2 , and M phases. The G 1 /G0 and S transition is a rate-limiting step in the cell cycle and represents the restriction point of the cycle [1]. G 2 /M phase is important for cell multiplication. The basic migratory cycle includes extension of a protrusion edge of a cell, formation of stable attachments near the leading edge of the protrusion, translocation of the cell body forward, and the release of adhesion molecule. All these steps require arrangement of actin cytoskeleton. Small GTPases of the Rho family are key regulators of these cytoskeletal dynamics. Rac-1, Rho-A, and Cdc-42 of Rho family GTPases are required for cell lamellipodial protrusions and activation of wave complex which provides force to cell migration and cell polarity establishment [2]. Like GTPase, cyclin-dependent kinases 1 and 2 are important for cell cycle control [3]. Wound healing requires both migration and proliferation of many cell types like neutrophils, fibroblasts, endothelial cells, and keratinocytes. Fibroblasts play important role in the process of wound healing and maintenance of epidermis dynamics with involvement of Rho-GTPase-dependent activation of basic fibroblast growth factor (bFGF) and collagen. This in turn leads to the activation of Rho-A, thereby facilitating both migration and proliferation of fibroblasts during the process of wound healing [4]. An understanding of the mechanisms that regulate the cell migration and proliferation of dermal fibroblasts cells by a natural compound could be beneficial in devising novel therapies to regulate fibrosis and wound contraction to ultimately improve the wound healing process.
Hyuganatsu, Citrus tamurana Hort. ex Tanaka, is one of the predominant citrus crops of Miyazaki, Japan. In recent years, this crop has increased the commercial value especially in food industries. Traditionally the citrus fruit has been used as a supplement to increase digestion and appetite, relieve flatulence and abdominal distension, and help in respiratory difficulties and also in the prevention of cough. Hyuganatsu peel extract (HE) has been reported to inhibit cytochrome P450 3A [5], suppress midazolam 1-hydroxylase activity of human CYP 3A [6] and inhibit hyaluronidase activity [7]. Furthermore, we have tested the efficacy of water soluble extract of Hyuganatsu extract in suppressing bone loss in ovariectomised rats [8]. However, whether it facilitates the process of in vitro wound healing and has a beneficial effect on the proliferation and migration of fibroblast cells remains to be explored. Therefore, in the present investigation, we tested the efficacy of HE on human fibroblast cell migration and proliferation and the associated cell cycle pattern and expressions of cell cycle regulatory pathways. Resources Bank (HRSBB, Osaka, Japan) and cultured in type-1 collagen coated plates (CELLCOAT, Greiner Bio-One, Germany). Cells were maintained in MEM with glutamine and 5% FBS in 10 cm culture plates. Cells were maintained in antibiotic cocktail at 37 ∘ C in a humidified incubator with an atmosphere of 95% air and 5% CO 2 . Cells at passages 2-5 were used for the experiments. All experiments were carried out in FBS deprived MEM + condition. HE was dissolved in sterilized water, sonicated, filtered, and sterilized through 20 M filter caps (Millex, Millipore Corp., Billerica, MA, USA), and stored at −20 ∘ C till further use.

In Vitro Wound
Healing Assay. TIG-119 cells were grown in 6-well plates at a density of 3 × 10 6 /mL, and a small linear scratch was created in the confluent monolayer by gently scraping with sterile cell scrapper as per standard methods [10]. Cells were extensively rinsed with medium to remove cellular debris before treating with different concentrations (0, 0.1, 0.25, 0.5, 0.75, and 1.0 mg/mL) of HE in FBS deprived condition. A positive control, prostaglandin I2 (PG12) analogue, and beraprost sodium (Kaken Pharmaceuticals, Co., Fukuoka, Japan) were used separately to judge the rate of cell migration. Twenty-four hours later, images of the migrated cells were taken using digital camera (Nikon, Tokyo, Japan), connected to the inverted microscope (Nikon, TMS-F, Japan), and analyzed by image analysis software (Image J, National Institutes of Health, Bethesda, MD, USA). Extent of wound healing was determined by the distance traversed by cells migrating into the denuded area. Representative data is cumulative of three independent experiments.

Cell Cycle Analysis by Flow Cytometer.
Fibroblasts cells (TIG-119) were treated with different concentrations of HE (0, 0.1, 0.25, 0.5, 0.75, and 1.0 mg/mL) for 24 h and fixed in cold 70% ethanol at 4 ∘ C. Cells were stored in the fixative at −20 ∘ C for 1 h. Following fixation, cells were centrifuged at 800 ×g for 5 min, resuspended in phosphate-citrate (PC) buffer at room temperature for 30 min, and again centrifuged at 1000 ×g for 5 min. The cells were resuspended in 800 L PBS before incubation in 100 L each of 100 g/mL RNase A and 0.01% propidium iodide. Flow cytometry analysis was performed after 30 min using Guava Cell Cycle Assay Mini Flow Cytometer (Millipore, Billerica, MA, USA).

Semiquantitative Real-Time PCR.
Total RNA was isolated from HE-treated groups using one step RNA isolation kit (ZyGEM Corp., Hamilton, New Zealand), and cDNA was prepared using Transcription High Fidelity cDNA synthesis kit (Roche Diagnostics, Indianapolis, IN, USA). RT-PCR was carried out as per standard procedures using ReverTra Ace enzyme (Toyobo, Tokyo, Japan).

Translation and Transcription Inhibition
Assays. Sub confluent cells were pretreated for 2 h with 10 g/mL of the translational inhibitor, cycloheximide (CHX) in serum-free medium. The cells were then treated with or without HE (0.75 mg/mL) and incubated further for 6 h. Total RNA was isolated, and expression levels of Cdk-1 and Cdk-2 were measured with -actin as internal control. DRB, a transcriptional inhibitor, was used to inhibit the rate of transcription. Cells were pretreated with 10 g/mL of DRB for 2 h in serum-free medium to prevent new DNA synthesis and treated with HE (0.75 mg/mL). Zero h represents the time of HE addition. Total RNA was isolated at various time intervals (0, 2, 4, 6, and 8 h), and Cdk-1 and Cdk-2 mRNA expression levels were measured against -actin.

Statistical Analysis.
Each experiment was carried out in three independent sets. Mean values and standard deviation were calculated. The Kruskal-Wallis test to compare more than two groups was used to judge the statistical significance. Statistically significant values were set at the level of < 0.05.

Results and Discussion
In this study, we used human skin fibroblasts (TIG-119) to investigate the effect of HE on the patterns of cellular crude nature of the test sample. This study supports several other investigations that reported that natural antioxidants increased the number of oral fibroblast [11] and endothelial cells [12] and modulated the growth of endometrial stromal cells [13].

HE Induces G 2 /M Stage of Cell Cycle.
Functional end point of fibroblast cell division and proliferation is cell migration [3] into wounded area [11]. An established in vitro scratch assay model was used to quantitatively define human skin fibroblast migration in a monolayer cell model by using NIH Image J software analysis. As depicted in Figure 3, HE dose dependently increased the rate of migration into wounded area up to the concentration of 0.75 mg/mL. The rate of cell migration was similar to that observed in the presence of beraprost, a potent cell migration inducer. Therefore it is proposed that HE is noncytostatic with proproliferative capacity to induce fibroblast cell migration.
Cell proliferation and migration are hallmarks of cell division. DNA duplication is the key step in cell division and it is controlled by different stages: G 1 /0, G 2 /M, and S phases of cell cycle [14]. Subsequently, we studied if HE treatment affected different stages of cell cycle. As seen in Figure 4, G 2 /M phases of cell cycle were dose dependently increased upon HE treatment with highest value obtained with 0.75 mg/mL treatment. As depicted in Figure 4(b), there were no significant differences in percentage of DNA count at G 2 /M phase (21. 2, 9.7, 17.6, 16.2, 10.1, and 16.3) and S phase (10.2, 12.4, 3.9, 2.5, 2.3, and 0.7) between different treatment groups, while DNA content in G1/0 phase (50. 2, 53.3, 59.7, 82.1, 89.4, and 63.3) varied significantly. This shows that HE does not arrest cells in G 2 /M phase but induces the initiation of DNA synthesis. Several lines of evidence support a molecular mechanism in the response to natural compounds stimulation that does arrest the G 1 /0 phases, and increases G 2 /M phase in fibroblast [15,16].

HE Acts Differentially on Rho Family GTPase and Cdk.
Fibroblast cell proliferation and migration phenomena are principally governed by Rho family GTPase like Rac-1, Rho-A, and Cdc-42 [4]. The cell cycle phases are coordinated by the expression and/or activation of regulatory proteins, like cyclins (e.g., cyclin A, D, and E), cyclin-dependent kinases (Cdk) mainly Cdk-1 and -2, and Cdk inhibitors. Both cyclins and cyclin-dependent kinases have also been implicated in the formation of actin cytoskeleton in mammalian fibroblast cells [17]. In order to gain an insight into the mechanisms of HE action, we studied the expression of Rac-1, Rho-A, Cdc-42, and Cdk-1 and Cdk-2 mRNA by RT-PCR assay and WB analysis. Cells exposed to 0.1, 0.25, 0.5, and 0.75 mg/mL. HE showed concomitant increase in both mRNA and protein levels of Rac-1, Rho-A, and Cdc-42 ( Figure 5(a)). Cyclindependent kinases have been identified as key proteins in the G 2 /M transition that help maintain steady-state level of M phase through inhibition of PP 2A/B558 [18]. Molecular mechanisms of mammalian cell migration were first revealed in fibroblasts where Rho-A, Rac-1, and Cdc-42 facilitate the multistep process including the establishment and maintenance of polarity, formation of actin-rich protrusions, remodeling of adhesive contacts, and generation of force. Our results revealed that HE (0.75 mg/mL) induces levels of Rho-A, Rac-1, and Cdc-42 ( Figure 5(a)) and helps in cell migrations as shown in Figure 3. Results also showed that mRNA levels of Cdk-1 and -2 increased significantly ( Figure 5(a)). However HE did not induce Cdk-1 & -2 protein levels ( Figures 5(a) and 5(b)). This observation leads us to hypothesie that HE extract differentially acts on mRNA and protein level working either at transcriptional or translation stage.
In order to prove the above hypothesis, we first treated subconfluent cells with HE (0.75 mg/mL) with/or without the translational inhibitor, CHX for 6 h, and analysed Cdk-1 and -2 mRNA expressions. As shown in Figure 6, HE alone (0.75 mg/mL) caused about 3-fold increases in both Cdk-1 and -2 mRNA activities; however, CHX abolished the effect of HE. Interestingly, CHX per se did not have any effect on Cdk-1 and -2 mRNAs. It is possible that induction of T Cdk-1 and Cdk-2 by HE could depend on some protein factor,   that is, inhibited by CHX treatment. We further checked the status of regulation at the transcription level. Subconfluent cells were conditioned for 2 h with DRB to inhibit ongoing transcription prior to treatment with HE (0.75 mg/mL) for varying time period. Figure 7 reveals the influence of DRB on Cdk-1 & -2 mRNA expression. The mRNA levels in both control as well as HE-treated group diminished with time course. HE failed to overexpress both Cdk-1 and -2 mRNA in the event of transcription inhibition. This implied that ongoing transcription was necessary for the HE action on Cdk-1 and -2 expressions.
The cyclin-E-dependent kinase Cdk-2 plays a crucial role in cell cycle progression, and the activity of Cdk-2 has been shown to be adhesion dependent and also correlates with mitogenic activity [19]. Taking into consideration the translation and transcription inhibition assays, we conclude that Cdk-1 and -2 regulations by HE are governed at transcription stage where de novo protein synthesis is required.

Summary
The evidence reported in the current study, combined with the relative safety of HE, suggests that HE has the potential to be developed as a new non-toxic nutraceutical agent for treating skin disorders. The results reported here should stimulate further research into identifying the active molecule which induces the coordinated cell migration and proliferation 8 BioMed Research International response in wounded tissues and thereby promotes a better understanding of this fundamental homeostatic process.