extfieldsofstudy: Chemistry Medicine

text:
K+ channels and cell cycle progression in tumor cells

K+ ions play a major role in many cellular processes. The deregulation of K+ signaling is associated with a variety of diseases such as hypertension, atherosclerosis, or diabetes. K+ ions are important for setting the membrane potential, the driving force for Ca2+ influx, and regulate volume of growing cells. Moreover, it is increasingly recognized that K+ channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently of ion fluxes. In cancer, aberrant expression, regulation and/or sublocalization of K+ channels can alter the downstream signals that converge on the cell cycle machinery. Various K+ channels are involved in cell cycle progression and are needed only at particular stages of the cell cycle. Consistent with this idea, the expression of Eag1 and HERG channels fluctuate along the cell cycle. Despite of acquired knowledge, our understanding of K+ channels functioning in cancer cells requires further studies. These include identifying the molecular mechanisms controlling the cell cycle machinery. By understanding how K+ channels regulate cell cycle progression in cancer cells, we will gain insights into how cancer cells subvert the need for K+ signal and its downstream targets to proliferate.

INTRODUCTION
Cell homeostasis requires a delicate balance between formation of new cells by cell proliferation and their elimination by apoptosis. Cell proliferation involves, at some point, activation of Cl − , K + , aquaporin, and Ca 2+ channels (Pardo, 2004;Lang et al., 2007;Lehen'kyi et al., 2007). As the respective channel inhibitors have been reported to interfere with cell proliferation, the channels appear to play an active role in the machinery triggering cell entry and progression through the cell cycle (Wonderlin and Strobl, 1996;Ouadid-Ahidouch and Ahidouch, 2008;Becchetti, 2011).
Perturbations in the cell cycle results in unlimited proliferation and confers the apoptosis resistance leading to cancer development Barbacid, 2001, 2009). The progression of cells through the cell cycle is regulated by different cyclin/Cyclin-Dependent Kinase (CDKs) complexes. The cyclin-CDK complexes govern a linear progression of the events that lead cells from a resting state (G0), to growth phase (G1), DNA replication phase (S), and finally to cell division (M). Cyclins D (D1, D2, and D3) and their associated CDKs (4/6) are important during the G1 phase (Caldon et al., 2006). Cyclins E and A, along with their partner, CDK2, are important for the G1/S phase transition and S-phase progression, respectively, and cyclins A and B (with Cdc2) are important for entry of the cells into the M phase (Schwartz and Shah, 2005). The activity of the cyclin/CDK complex can be inhibited by Cyclin-Dependent Kinase inhibitors (CDKi) such as p21 WAF1/Cip1 and p27 kip1 (Besson et al., 2008).
Over the past two decades, ion channels have been identified as important contributors to cell proliferation of normal and cancerous cells (for review see Wonderlin and Strobl, 1996;Kunzelmann, 2005). Otherwise, K + channels are thought to be important for setting the membrane potential and the driving force for Ca 2+ influx and both mechanisms are considered crucial for volume regulation of growing cells (Nilius and Wohlrab, 1992;Kunzelmann, 2005;Lang et al., 2007;Ouadid-Ahidouch and Ahidouch, 2008). Moreover, it is increasingly recognized that K + channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently from ion fluxes (Hegle et al., 2006;Kaczmarek, 2006;Glassmeier et al., 2012).
Although the K + channels contribute to regulation of different cell cycle checkpoints in cancer cells, no direct evidence about the mechanism at issue has been provided concerning how they control the cell cycle machinery, particularly in regard to CDKi and/or cyclin expression (Evan and Vousden, 2001). This review focuses on the regulation of proliferation by K + channelmediated signals and discusses how these signals may influence the cell cycle progression in tumor cells.

K + CHANNELS AND CELL PROLIFERATION
K + channels belong to a large and heterogeneous group of ion channels. The first K + currents were described by Hodgkin and Huxley in the early 50s during their work on the identification of ionic species responsible for the propagation of the action potential in the squid giant axon. Today, several types of K + channels have been identified by molecular cloning. They are responsible for the passive diffusion of K + ion across the plasma membrane and are detectable in both excitable and non-excitable cells. K + channels play a major role in several physiological functions such as synaptic transmission, muscle contraction, release of hormones, such as insulin (for review see Sandhiya and Dkhar, 2009), or in proliferation, apoptosis, migration, invasion, and angiogenesis (Pardo, 2004;Cherubini et al., 2005;Ouadid-Ahidouch and Ahidouch, 2008;Schwab et al., 2008;Becchetti, 2011;Cuddapah and Sontheimer, 2011;Jehle et al., 2011;Girault et al., 2012). The physiological importance of K + channels is undoubtedly underscored considering the increasing number of genetic diseases associated with them. Indeed, Kv gene mutations trigger episodic ataxia, long QT syndrome, pancreatic diseases, and epilepsy (Ashcroft and Gribble, 2000;Goonetilleke and Quayle, 2012).
The early studies that have linked K + channels to the proliferative processes were carried on T lymphocytes (DeCoursey et al., 1984;Matteson and Deutsch, 1984). In cancer, the first works were performed on melanoma cells, neuroblastoma, breast, lung, and bladder cancer cells (Rouzaire-Dubois and Dubois, 1990;Lepple-Wienhues et al., 1996;Wonderlin and Strobl, 1996;Ouadid-Ahidouch and Ahidouch, 2008). Since then, a wide variety of K + channels has been shown to be implicated in the regulation of proliferation of myeloid leukemia, prostate, colon, neck, and head cancer, lymphoma and hepatocarcinoma cells (Zhou et al., 2003;Conti, 2004;Pardo, 2004;Wang, 2004;Kunzelmann, 2005;Wang et al., 2007a;Arcangeli et al., 2009;Jehle et al., 2011;Lehen'kyi et al., 2011). These data are based on the fact that, in vitro, genetic or pharmacological inhibition of K + channels inhibits the growth of cancer cells. Moreover, K + channel openers can also be responsible for an increase in proliferation rate. Indeed, minoxidil or cromakalim increased activity of K ATP channels inducing an increase in DNA synthesis (Malhi et al., 2000). KCa3.1 channels activator, 1-EBIO, increased prostate cancer cell proliferation (Parihar et al., 2003), but diminished the proliferative effect on keratinocytes (Koegel et al., 2003).
One of the most accredited models is based on the involvement of K + channels in the regulation of membrane potential, which drives cancer cells into certain phases of the cell cycle. In cancer cells, it has been shown that cells in the early G 1 phase are depolarized; however, they are hyperpolarized during the progression through G 1 and into the S phase (Wang et al., 1998). Inhibition of K + channels or blocking cells in G1 phase by serum starvation is accompanied by membrane depolarization (Nilius and Wohlrab, 1992;Wonderlin et al., 1995;Wang et al., 1998;Ouadid-Ahidouch et al., 2001). Similarly, membrane depolarization caused by an increase in the concentration of extracellular K + mimics the effects of K + channel blockers (Freedman et al., 1992).
Many K + channels have been implicated in the regulation of the membrane potential, (for review see: Wonderlin and Strobl, 1996;Pardo, 2004;Kunzelmann, 2005;Ouadid-Ahidouch and Ahidouch, 2008;Becchetti, 2011). In some cases, the membrane potential of cycling cells is regulated by the ratio of different K + channel isoforms. For example, in neuroblastoma, the membrane potential can be controlled by Kv11.1 and blocking this channel inhibits mitosis (Arcangeli et al., 1995;Crociani et al., 2003;Becchetti, 2011). In these cells, the membrane potential tends to depolarize during the S phase. The effect is caused by oscillation of the expression balance of the full-length Kv11.1a isoform and the N-deleted Kv11.1b (Crociani et al., 2003). Indeed, the truncated Kv11.1b form is up-regulated during the S phase, while the full-length Kv11.1a protein increases its expression on the plasma membrane during the G1 phase (Arcangeli et al., 1995). The increase of the ratio between Kv11.1b and Kv11.1 leads to the depolarization of the membrane potential occurring during S phase progression (Arcangeli et al., 1995).
Ca 2+ signaling appears to be required for progression through G1, the G1/S transition, and G2/M in several cell types (Kahl and Means, 2003;Roderick and Cook, 2008;Ding et al., 2010). The relationship between K + channels, membrane potential, and Ca 2+ influx has been first reported in T cells by Cahalan's group. Indeed, the authors proposed that the membrane hyperpolarization generated by the activation of K + channels increases the driving force for Ca 2+ influx (Hess et al., 1993;Lewis and Cahalan, 1995), which in turn, activates Ca 2+ -dependent transcriptional factors leading to expression of cell cycle regulatory proteins, such as cyclins, CDKs, and the inhibition of CDKi expression (Nilius and Wohlrab, 1992;Yao and Kwan, 1999;Ouadid-Ahidouch et al., 2004;Zhanping et al., 2007;Borowiec et al., 2011). The inhibitors of voltage-gated K + channels block the Ca 2+ influx and the cell proliferation (Yao and Kwan, 1999;Ouadid-Ahidouch et al., 2004;Zhanping et al., 2007).
In agreement with these general considerations, in prostate LNCaP cells, a member of Transient Receptor Potential (TRP) channel family, TRPV6 was identified as a major provider of passive calcium influx in response to hyperpolarization associated with activation of the intermediate-conductance Ca 2+ -activated K + channel (KCa3.1) (Lallet-Daher et al., 2009). The same team showed that calcium entry through TRPV6 channel in these cells induced a subsequent downstream activation of Nuclear Factor Activated T cell (NFAT) leading to cell proliferation (Lehen'kyi et al., 2007). In breast cancer cells, it was demonstrated that Eag1 (Kv10.1) K + channel function is required for controlling the Ca 2+ entry trough Orai1 channels (Hammadi et al., 2012).
K + channels also control cellular proliferation by affecting cell volume (Rouzaire-Dubois et al., 2000). Alterations of cell volume require participation of ion transport across cell membrane and accordingly, Cl − and K + channels (Lang et al., 2007). Recently, aquaporin channels have emerged as regulators of cell cycle progression (Di Giusto et al., 2012). Cell volume regulation may favor maintenance of appropriate levels of critical cell cycle regulatory proteins that are necessary for controlling cell cycle progression through G1 to S or G2/M phases (Tao et al., 2008;Huang et al., 2012).
In addition to their ability to permit ions to cross the membrane, ion channels can also have non-conducting functions that enable them to interact with cell signaling pathways to directly regulate biochemical events (Kaczmarek, 2006). It has been demonstrated that expression of a mutant form of Eag1, that cannot conduct current, promotes proliferation of NIH3T3 fibroblasts and C2C12 myoblasts cells (Hegle et al., 2006). In contrast to the wild type Eag1, the no-conducting Eag1 is independent of serum and is unaffected by changes in extracellular Ca 2+ . Moreover, proliferation induced by Eag1 is unaffected by changes in extracellular Ca 2+ , suggesting that increased Ca 2+ influx is not an essential downstream component of Eag1induced signaling. The same authors reported that Eag1 is able to regulate cell proliferation in fibroblasts via activation of the p38 Mitogen-Activated Protein (MAP) kinase pathway but not extracellular signal-regulated kinase 1/2 (ERK1/2). Ectopic expression of pore-dead mutant Eag1, in CHO cells, promotes cell growth in vitro and in vivo (xenografts) (Downie et al., 2008). Recently, the ion-conducting function of HERG1 has also proved to be important for cell growth of human small cell lung cancer cells (Glassmeier et al., 2012). Indeed, the knockdown of HERG1 inhibits cell proliferation, while its pharmacological inhibition by E4031 fails to affect cell proliferation (Glassmeier et al., 2012). Additionally, the expression of non-conducting mutant KCa3.1 induced HEK293 cells proliferation not by enhancing Ca 2+ entry but via a direct interaction with ERK1/2 and c-jun N-terminal kinase (JNK) signaling (Millership et al., 2011). Increasing number of reports show that certain K + channels interact with signaling molecules directly to regulate cellular signaling. Indeed, the N-and C-terminal domains of hEag1 interacts with calmodulin (Schönherr et al., 2000;Ziechner et al., 2006;Gonçalves and Stühmer, 2010), cortactin (Herrmann et al., 2012); KCa3.1 channels interact with ERK1/2 (Millership et al., 2011), and HERG1 channels with the adaptor protein 14-3-3 (Kagan et al., 2002), Src tyrosine kinase (Cayabyab and Schlichter, 2002), and the TNF-α receptor . In addition, HERG channel proteins have been shown to interact with integrins, thereby regulating cell survival, adhesion and migration (Arcangeli and Becchetti, 2006;Arcangeli, 2011).
The mechanisms that allow K + channels to regulate cell growth in cancer cell lines appear to be different from the ones in normal cells. It has been reported that cancer cells express several K + channel isoforms that may be physiologically different as compared to a wild type channel. For example, proliferation of neuroblastoma cells is regulated by an oscillation balance of expression of the full-length HERG 1a (Kv11.1a) isoform and the N-deleted HERG 1b (Kv11.1b) (Crociani et al., 2003). It should be also noted that the effects of K + channels on cell proliferation can involve their trafficking to the micro-domains within the cell.
It follows from the foregoing that the K + channels influence cell proliferation through as many mechanisms as the number of their families and correspondent isoforms. That is undoubtedly dependent on intrinsic features of each cell type and the isoforms expressed.

K + CHANNEL ACTIVITY DURING THE CELL CYCLE
A direct link between channel activity and particular stages of the cell cycle has been reported. For example, in HeLa cells, the K + current-density increases during M and G1 phases (Takahashi et al., 1993), in unfertilized mouse oocytes, a large-conductance, voltage-activated K + channel (BKCa, 240 pS), is active throughout M and G1 phases, and switches off during the G1-to-S transition (Day et al., 1993). In Xenopus oocytes, the expressed rat Eag (rEag1) rEag1 displays decrease in current-density in meiotic phase induced by progesterone or by Mitosis promoting factor (Brüggemann et al., 1997). Furthermore, the partial synchronization of cells in G1 or M phases greatly increases the current blockade by intracellular Na + and Cs + (Pardo et al., 1998). In cancer cells, the K + channels activity has been also found to be cell cycle-dependant. For example, the activity of Eag1 is at high rate in G1 phase and decreases when cells enter S phase (Ouadid-Ahidouch et al., 2004), and in neuroblastoma cells, HERG current activity has been shown to be cell cycle-dependent (Arcangeli et al., 1995).
The mechanisms linking the activity of each of these channels to the cell cycle appear to be different and include regulation by cytoskeletal elements (Camacho et al., 2000), the activation of cyclin-dependent kinase 1 (CDK1) cyclin B (Brüggemann et al., 1997), a cytoplasmic cell cycle that can run independently of the nuclear cell cycle (Day et al., 1998), or channel trafficking.
Studies on K + channels trafficking are increasingly emergent. It has been suggested that Eag1 turns over rapidly (8-12 h) at the cell surface (Weber et al., 2006). This process involves surface expression followed by constitutive internalization and degradation in lysosomes (Kohl et al., 2011). Indeed, the Eag1 trafficking has been reported to be regulated by several proteins including cortactin, rabaptin-5 and epsin. Depletion of cortactin decreases the number of functional Eag1 channels without altering their open probability or conductance (Herrmann et al., 2012). These authors suggest that cortactin connects Eag1 to the cytoskeleton. Moreover, knockdown of rabaptin-5 reduces recycling rates of Eag1 and leads to a reduction of Eag1 current-density . Eag1 also interacts with epsin that modulates its gating in rat brain (Piros et al., 1999). In view of these studies, the expression of K + channels during the cell could likely due to their trafficking, although the involvement of this particular pathway in cell proliferation is still not proven.

MOLECULAR MECHANISMS UNDERNEATH CELL CYCLE REGULATION BY K + CHANNELS (SEE TABLE 1 AND FIGURE 1)
Aberrantly expressed cell cycle-related cyclins are highly associated with several cancer types including breast cancer (for review Williams and Stoeber, 1996). As such, cyclin D1 is overexpressed in ∼50% of breast cancers (Alle et al., 1998). Cyclin E is overexpressed in 30% of breast tumors (Wang and Shao, 2006), and the elevation of cyclin E is correlated with low levels of p27 Kip1 . A decrease in the expression of p27 Kip1 is strongly detected in breast cancer tissues (for review, Alkarain et al., 2004). Further genetic abnormalities also affect other CDKi, such as p16 INK4a , p15 INK4b , and p57 Kip2 (for review, Williams and Stoeber, 1996). These observations led to consider the cell cycle regulators as potential targets for selective inhibition in the treatment of cancer (Stone et al., 2012). The studies conducted so far show that the modulation of these key proteins of the cell cycle correlates with tumor development. Thus, it is clear that mitogenic factors capable of modulating their expression positively can intensify the process of carcinogenesis.
Despite K + channel blockers modulate several proliferative signaling pathways evidence for a direct mechanistic link between K + channels and cell cycle proteins remains sparse to date. Two cell-cycle blocks are often observed when K + channels are inhibited: at G1 (early G1, late G1, G1/S transition) and at G2/M block with or without changes in the proportion of the cells entering S-phase. The G1 block is associated with a reduction of the expression of cyclins (D and E), CDKs (4 and 2), pRb activation, and overexpression of CDKi (p21 Waf1/Cip1 and p27 kip1 ), while the block in G2/M is associated with a reduction of cyclin B1 expression or changes in Cdc2 (CDK1) phosphorylation. The first studies used K + channels pharmacological inhibitors such as TetraEthyl Ammonium (TEA) and 4-Amino-Pyridine (4-AP) to demonstrate that inhibition of the two K + channels (Kv1.3 and Kv1.5) resulted in cell cycle arrest at G1 in rat oligodendrocyte precursors (Ghiani et al., 1999;Chittajallu et al., 2002). The authors proposed that changes in the membrane potential (depolarization) could activate a signaling pathway involving the p27 kip1 and p21 Waf1/Cip1 proteins expression. Similar results were obtained when the membrane potential was depolarized by increasing extracellular K + (Renaudo et al., 2004).
In small cell lung cancer (SCLC, NCI-H209, and NCI-H146) and leukemic (Jurkat) cell lines, inhibition of K + channels has also been implicated in upregulation of the p27 kip1 protein and a reduction in cyclin. A expression resulting in cell arrest in G1/S phase transition (Renaudo et al., 2004). Athough the mechanisms, by which K + channels regulate cell cycle in these cells remain undetermined, the authors suggested involvement of cytoskeleton rearrangements due to cell volume changes.
A detailed mechanism underlying the role of Eag1 in the cell cycle was studied in MCF-7 breast cancer cells. In this work, mitogenic stimulation [using serum or Insulin Growth Factor 1, (IGF-1)] up-regulates Eag1 expression and activity (Borowiec et al., 2007). IGF-1 triggers cell cycle progression by increasing expression of cyclin D1, cyclin E, CDK4, CDK2, and phosphorylation of pRb (Dufourny et al., 1997;Borowiec et al., 2011). Inhibition of Eag1 by astemizole or by siRNA induced a decrease in cyclin D1 expression along with a strong decrease of pRb phosphorylation and an arrest of the cells in G1 phase of cell cycle. The effect of Eag1 inhibition was accompanied by a decrease of cyclin E expression, the key regulator of the G1/S transition necessary for cell entry into S phase (Skelding et al., 2011). However, inhibition of Eag1 failed to modify the p21 Waf1/Cip1 , p27 Kip1 , and CDK4/2 expression.
Interestingly, when Eag1 is inhibited, the cyclin D1 level is below its level in serum-deprived cells, the condition known to synchronize cells in G1 phase (Borowiec et al., 2011). It was thus suggested that Eag1 control of the cell cycle could be upstream of the G1 phase synchronization by serum deprivation. Indeed, the cell cycle arrest site under Eag1 inhibition is both upstream of that obtained by serum-deprivation, and downstream of that induced by lovastatin, which synchronizes the cells in the early G1 phase (Borowiec et al., 2011). Altogether, these results demonstrate that Eag1 activation is positioned upstream of the upregulation of both cyclins D1 and E expression.
It has been reported recently that the isoform 2 of Eag (Eag2) is essential for mitotic entry of medulloblastoma (MB) cells (Huang et al., 2012). Indeed, the authors observed a correlation between temporal Eag2 translocation to plasma membrane and mitotic entry from the late G2 phase concurrent with an increase of outward K + current during mitosis. Moreover, Eag2 knockdown accumulates cells in G2/M in association with a strong decrease of cyclin B1 (essential for G2/M transition), but fails to affect the expression of cyclin A, cyclin D1, cyclin E, and CDK1 (Huang et al., 2012). The G2/M arrest is due to an alteration in cell volume control that is linked to hyperactivation of p38 MAPK pathway without any effect on p53 expression. It has been then hypothesized that Eag2 regulates cell volume during the MB cell cycle progression (Habela and Sontheimer, 2007;Boucrot and Kirchhausen, 2008).
Ca 2+ -activated K + channels (KCa) also regulate cell cycle progression. KCa3.1 is one of the KCa channels that regulate proliferation and migration of cancer cells (Ouadid-Ahidouch et al., 2004;Faouzi et al., 2010;Catacuzzeno et al., 2012). KCa3.1 controls G1 (mainly late G1) phase, G1/S transition, and G2/M phase. In breast, prostate and endometrial cancers, KCa3.1 regulates G1 and G1/S transition (Ouadid-Ahidouch et al., 2004;Wang et al., 2007b;Lallet-Daher et al., 2009). Pharmacological or genetic blockade of KCa3.1 increases p21 Waf1/Cip1 expression and decreases the expression of cyclin E. According to the "potential membrane model," activation of K + channels amplifies the Ca 2+ signals by hyperpolarizing the membrane potential, thus increasing driving force for Ca 2+ influx, which, in turn, activates Ca 2+dependent transcriptional factors leading to expression of G1 cyclins and CDK proteins (Roderick and Cook, 2008). Consistent with this notion, it has been shown that TRPV6 may be the major provider of passive Ca 2+ influx in response to the hyperpolarization associated with KCa3.1 channels activation in prostate cancer cell line (Lallet-Daher et al., 2009). In breast cancer cells, it has been demonstrated that Eag1, by regulating membrane potential, controls Ca 2+ entry through Orai1 channels (Hammadi et al., 2012).
KCa3.1 channels also control the cell volume and regulate G1/S transition and G2/M phases (Tao et al., 2008;Lai et al., 2011). In mouse mesenchymal stem cells, KCa3.1 channels associated with Cl − -volume channels regulate cell cycle progression by modulating cyclin D1 and cyclin E expression (Tao et al., 2008). However, in human colon cancer cells (LoVo), their inhibition accumulates cells in G2/M phase and increases the phosphorylation of Cdc2 (Lai et al., 2011).

CONCLUSIONS
In summary, K + channels are often overexpressed in tumor cells and regulate proliferation. They are needed to control specific checkpoints in the cell cycle progression (early G1, the G1/S and G2/M transitions). Accordingly, several K + channels exhibit a cell-cycle-dependent expression and activity. In addition, cell cycle progression is often accompanied by oscillations of Cl − channel expression and Ca 2+ signals that regulate cell proliferation. Recent evidence shows the involvement of aquaporin in cell proliferation. The links between K + channels and cell-cycle machinery (cyclins, CDKs, and CDKi) start to emerge (Figure 1). However, we are still far from full understanding the complex mechanistic link between channel expression/activity FIGURE 1 | Schematic illustration of the role of K + channels during cell cycle progression and proliferation.
(1) Membrane potential model: during progression into G1 phase, the membrane potential becomes hyperpolarized relative to the normal resting potential. The hyperpolarization relates to Kv channels activation (for example Kv1.3, Eag1) in early G1 and to Ca 2+− activated K + channels (for example KCa3.1) activation in late G1 and/or G1/S transition. Multiple growth factors in serum have been well-described to play an important role in initiating G1 progression to the S phase of the cell cycle, in which Ca 2+ influx is a major determinant in serum-induced DNA synthesis. Growth factors (for example IGF-1) binding to the receptor tyrosine kinases [RTKs; for example, Insulin growth factor receptor (IGF-1R)] can activate effectors [Extracellular signal-Regulated Kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)] that increase the expression and the activity of K + channels inducing a hyperpolarization of the membrane potential (MP). Moreover, RTKs also activate phospholipase C (PLCγ) to promote the generation of inositol-1,4,5-trisphosphate (InsP3) and the release of Ca 2+ from the endoplasmic reticulum (ER) into the cytosol. Ca 2+ enters across plasma membrane by store-operated capacitive Ca 2+ entry (SOCE) through the ORAI1 (Hammadi et al., 2012) or ORAI3 (Faouzi et al., 2011) channels, through TRP Ca 2+ channels (for example TRPC1, El Hiani et al., 2009), or via the constitutively active TRPV6. The Ca 2+ entry, in turn, regulates the activity and/or the expression of Ca 2+ -activated K + channels (for example KCa3.1), which maintains the hyperpolarization promoting a significant Ca 2+ -entry. The increase in [Ca 2+ ]i triggers the activation of Ca 2+ -dependent signaling enzymes that may act by regulating the expression or activity of the transcription factors (TF), such as FOS, JUN, NFAT, C-MYC leading to the expression of cyclins and CDKs and the inhibition of the CDK inhibitor proteins (p27 KIP1 and p21 waf1/cip1 ). (2) Volume control: K + channels in association with chloride channels regulate cell cycle progression by controlling the cell volume. For example, Eag2 channels control M phase by regulating the expression of cyclin B1 through the p38 MAP Kinase pathway. (3) Non-conducting roles: K + channels may also promote cell proliferation independently of their ion permeation function. For example: Eag1 and KCa3.1 induce cell proliferation by (direct or indirect) interaction with MAP kinase signaling pathways (p38 for Eag1, ERK1/2, and JNK for KCa3.1). MP: membrane potential, (vol. CTRL): volume control, (PM): plasma membrane. and signaling in proliferating cells. The detailed understanding of the role of K + channels and their connection with Ca 2+ signals in regulation of cell cycle proteins and/or transcription factors will offer significant opportunities to develop more specific tools for cancer therapy.

ACKNOWLEDGMENTS
This study was supported by the Region Picardie, by the Ministère de l'Education Nationale (France), and la Ligue Nationale Contre le Cancer. We thank Pr Irina Korichneva for critical reading of the manuscript.


extfieldsofstudy: Medicine Biology

text:
Vector-borne helminths of dogs and humans in Europe

Presently, 45% of the total human population of Europe, as well as their domestic and companion animals, are exposed to the risk of vector-borne helminths (VBH) causing diseases. A plethora of intrinsic biological and extrinsic factors affect the relationship among helminths, vectors and animal hosts, in a constantly changing environment. Although canine dirofilarioses by Dirofilaria immitis and Dirofilaria repens are key examples of the success of VBH spreading into non-endemic areas, another example is represented by Thelazia callipaeda eyeworm, an emergent pathogen of dogs, cats and humans in several regions of Europe. The recent finding of Onchocerca lupi causing canine and human infestation in Europe and overseas renders the picture of VBH even more complicated. Similarly, tick-transmitted filarioids of the genus Cercopithifilaria infesting the skin of dogs were recently shown to be widespread in Europe. Although for most of the VBH above there is an increasing accumulation of research data on their distribution at national level, the overall impact of the diseases they cause in dogs and humans is not fully recognised in many aspects. This review investigates the reasons underlying the increasing trend in distribution of VBH in Europe and discusses the diagnostic and control strategies currently available. In addition, this article provides the authors’ opinion on some topics related to VBH that would deserve further scientific investigation.

Introduction
A large number of vector-borne helminths (VBH) are prevalent in Europe, and some of them are of growing importance due to the significant level of disease they cause in dogs and humans [1][2][3]. Presently, 45% of the total human population of Europe, as well as their domestic and companion animals, are exposed to the risk of VBH [4]. A complex range of intrinsic biological factors (e.g., vectorial capacity, biting rates), extrinsic and environmental factors (e.g., climate, population movements and trade), affects the interactions between parasitic helminths, vectors and animals, including humans, rendering investigations on VBH a complex task. Indeed, the spreading process of VBH in previously non-endemic geographical areas has been primarily associated with the biology and ecology of the arthropod vectors and their capability to establish transmission cycles, maintaining the infestation in populations of susceptible hosts. Since the beginning of the millennium, many vectors have been introduced into Europe as a consequence of human demographics (e.g., the growth of cities), international movement of people (travellers and refugees), the smuggling of wildlife, the trade of animals and goods, such as used tires and ornamental plants [5]. For example, human activities have initiated the spread of invasive mosquito species and vector-borne diseases, and on-going globalization and increases in mean temperature may greatly extend the magnitude of this process [4].
The present article is focused on major VBH infesting dogs and humans. Among this diverse group of pathogens, Dirofilaria immitis and Dirofilaria repens (Spirurida, Onchocercidae) are probably the best known. Indeed, D. immitis has a severe impact on veterinary medicine, because of the heartworm disease threatening dogs and cats, whereas D. repens, causing subcutaneous infestation in dogs, is the main agent of human dirofilariosis. Although Dirofilaria spp. above represent key examples of the success of this group of parasites in spreading into non-endemic areas, over the last ten years other zoonotic helminths, such as Thelazia callipaeda eyeworm (Spirurida, Thelaziidae) have been accounted as emergent VBH of animals and humans in several Europe regions [5]. In addition, the puzzle become even more complicated to solve by the recent finding of a little known filarioid of dogs, i.e. Onchocerca lupi (Spirurida, Onchocercidae), which causes canine and human infestation in Europe and overseas. This nematode primarily induces nodular lesions under the conjunctiva and sclera of dogs and its biology and actual distribution remain for many aspects unknown to science. Adults of the less known Acanthocheilonema reconditum and Acanthocheilonema dracunculoides are beneath the subcutaneous tissues of the limbs and back of dogs. Recently, tick-transmitted filarioids of the genus Cercopithifilaria infesting the skin of dogs were shown to be surprisingly distributed in canine populations of Europe [6].
Although for many of the VBH above there is an increasing accumulation of information about their distribution at national level, the overall impact of diseases they cause in dogs and humans is not fully recognised in many aspects. This review aims at investigating the main reasons underlying the increasing trend in distribution of the most important VBH in Europe and to discuss the diagnostic and control strategies currently available. In addition, this article provides the authors' opinion on some topics related to VBH that would deserve further scientific investigation.

Old and emerging VBH of dogs and humans in Europe
Dirofilarioses caused by filarioid nematodes of the genus Dirofilaria are transmitted at their third larval stage by bloodsucking mosquitoes primarily to dogs, although cases of human dirofilariosis are increasingly reported [1]. Adult D. immitis worms occur in the pulmonary arteries and right heart chambers, causing a severe condition, known as canine and feline heartworm disease, while D. repens is found mainly in subcutaneous tissues, causing subcutaneous dirofilariosis. Dirofilaria nematodes develop throughout five larval stages within the intermediate vector mosquito host (from embryo to infective L3 larva), and in the definitive vertebrate host (from L3 to the adult stage). The adult females of D. immitis and D. repens develop in 120-180 and 189-259 days, respectively, and release microfilariae into the blood of the definitive host [7]. The intermediate hosts are mosquitoes of the family Culicidae (e.g. Anopheles, Aedimorphus, Armigeres, Ochlerotatus, Stegomyia, Culex, Coquillettidia and Mansonia), with Aedimorphus vexans [Aedes vexans], Culex pipiens pipiens ( Figure 1) and Stegomyia albopicta [Ae. albopictus] (Figure 2) being implicated as the main vectors of these worms in Europe [8]. Dirofilaria repens is able to grow under laboratory conditions in the same mosquito species and at the same temperature and humidity as D. immitis, with similar developmental time, from the microfilarial stage to the infective larva [7].
Over the last 20 years, T. callipaeda has been repeatedly reported to infest the conjunctival sac of domestic (dogs and cats) and wild carnivores (e.g., foxes, wolves, beech martens and wild cats) in Europe [10]. Nowadays, this nematode is recognised as endemic in many European countries ( Figure 3) such as France [11,12], Switzerland [13], Spain [14] and Portugal [15] at the similar latitude range (between 39°and 46°N) than those of Asia (between 10°and 45°N for India and Japan, respectively) where the infestation seemed to be confined [16]. Indeed, for its geographical distribution (i.e., in the former Soviet Republics and in many far eastern countries including India, Thailand, China and Japan), this nematode has been known for a long time as "oriental eye-worm" [17]. Since the first evidence of its development in a "bizarre" drosophilid vector, Phortica variegata (Diptera, Drosophilidae, Steganinae) [18,19], the occurrence of this helminth seems to be on the rise, probably also due to the improved awareness of parasitologists and practitioners. Interestingly, cases of human thelaziosis in Europe have been diagnosed in north-western Italy, south-eastern France [20] and Spain [21].
Onchocerca lupi is an even less known VBH parasitizing the periocular tissues of dogs and cats which has been recognised as a valid species on morphological and molecular grounds [22]. This parasite has been found to infect dogs in southern (Greece, Portugal) and Central Europe (Germany, Hungary, Portugal, Switzerland) [23][24][25][26][27] (Figure 3) and the United States [28][29][30] where it was recently found also in cats [31]. It causes acute or chronic ocular disease, characterized by conjunctivitis, photophobia, lacrimation, ocular discharge and exophthalmia [32]. Unfortunately, the role played by dogs as reservoirs of O. lupi deserves to be assessed and knowledge on the biological vector of this infestation remains meagre [33].
A group of rather neglected filarioids belonging to the genus Cercopithifilaria parasitizing the skin of a range of host species [34] has also been recently studied in dogs [35] and three species (namely Cercopithifilaria sp.1, Cercopithifilaria sp.2 and Cercopithifilaria grassii) have been morphologically and molecularly differentiated [36]. In addition, for Cercopithifilaria sp.1, the competence of the brown dog tick, Rhipicephalus sanguineus, as intermediate host has been experimentally demonstrated [37] and field evidence supports their role as vectors of this filarioid [6]. Following the first retrieval of Cercopithifilaria sp.1 from a dog from Sicily, Italy, this filarioid was diagnosed in dogs from Spain, Greece and southern Italy (i.e., Apulia, Basilicata and Sicily regions) ( Figure 3), with prevalence rates reaching up to 21.6% [6]. While their pathogenicity to humans is unlikely, there is some evidence indicating the occurrence of skin alterations associated to the presence of larvae in the dermis of dogs [38].

The role of vectors of VBH in a changing environment
The dissemination of VBH in Europe has been primarily attributed to rapid geographic expansion of their vectors (e.g., invasive mosquitoes, zoophilic fruit flies) and/or increases in their population density. This results from the interaction of several factors, such as the availability of suitable hosts, the arthropod's adaptability to different environmental conditions, its feeding behaviour and host preferences [39][40][41]. There have also been extensive debates on the effects of climate change in Europe [42,43], since warmer climates could favour mosquito breeding and, along with higher air temperatures, shorten their extrinsic incubation periods as demonstrated for Stegomyia aegypti [Ae. aegypti] [44]. Indeed, projected increment in temperature will impact on insect vectors through broadening areas of colonization, invasion of new sites and, eventually, resulting in physiological changes and increased vector capacity. For example, climate change (e.g., increase in mean temperatures) has affected the mosquito abundance and their seasonal survival in many areas of Europe greatly impacting on the spread of filarial infestation [7]. Growing degree-day models (GDD), using wide or local scale temperature data, predicted the occurrence and seasonality of Dirofilaria spp. in different parts of the world. These models were based on the minimum threshold of 14°C for the development of Dirofilaria in their vector, the requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito [7]. Therefore, it was predicted that, due to global warming and raising of mean temperatures, most of the European countries will be suitable for Dirofilaria transmission, with a lengthening in the duration of the filarial transmission season [7].
In addition, several intrinsic factors linked to the specific mosquito vector species also impact on the distribution of VBH. For example, based on retrospective evidence, the expansion of dirofilariosis in Europe somehow matched the second introduction of St. albopicta (in 1990 in Italy) [45] but it was not before 2000-2002, when both D. immitis and D. repens were found in natural populations of Asian tiger mosquito in Italy [46]. Accordingly, the rapid spread of this vector species throughout the country likely broadened the dirofilariosis range to southern regions not previously infected [47] (Figure 4), although the same areas were inhabited by Cx. p. pipiens, which is considered the main vector of both Dirofilaria in Europe. The sympatric occurrence of both vectors, having diurnal and nocturnal biting activities, may enhance the risk of infestation to dogs and humans, thus increasing the vector-host contact and, eventually, the number of vectors which may carry filarioids in endemic areas throughout the day. Interestingly, over the past decades, Cx. p. pipiens has changed its endophagic and antropophagic behaviour in Central and North Europe [4] where actually it also searches for human blood outdoors, as it happens in southern parts of the continent. This pattern also overlaps with the spread of canine D. immitis and D. repens infestation in central and north-eastern countries (e.g. south of Switzerland, Czech Republic, Hungary, Serbia and Slovak Republic) [7,[48][49][50][51][52] (Figures 5,6). Black flies may play a role in the transmission of O. lupi in dogs and humans, no convincing scientific evidence in this regard has been produced so far [33]. One of the suspected vectors of O. lupi, Simulium reptans, is present in areas where the cases of dog ocular onchocerciasis have also been reported (i.e., Portugal, Switzerland, Germany and Hungary) [33]. Until about 50 years ago, S. reptans was the dominant black fly in the middle part of Danube valley, whereas today it is extremely rare in this area and it moved southwards down into the Balkan Peninsula [54]. However, in addition biting midges (Diptera, Ceratopogonidae) feeding on a wide range of hosts (humans, livestock and other mammals, amphibians, and birds) might be implicated in the transmission of O. lupi since some species of Culicoides have been involved in transmission of Onchocerca cervicalis and Onchocerca gutturosa to horses and cattle, respectively, in Europe [55].
In recent years, the use of the Geographic Information System (GIS) and predictive model algorithms provided important practical contributions to the investigation of the spatial component of the epidemiology of infectious diseases [56], including vector-borne diseases [57,58]. Moreover, the collection of georeferenced epidemiological data can also be useful for disease cluster identification and geostatistical analyses. For example, regional climate model scenarios coupled with high-resolution  observations shows that during the 1960-1980s southern France, northern Italy, the northern coast of Spain, the eastern coast of the Adriatic Sea and western Turkey were climatically suitable areas for the establishment of the invasive Asian tiger mosquito, St. albopicta. Over the last two decades, climate conditions have become more suitable for the Asian tiger mosquito over Benelux, western Germany and the Balkans, while they have become less suitable over southern Spain. Similar trends are likely to be observed in the future, with an increased risk simulated over northern Europe and slightly decreased risk over southern Europe where drier and warmer summers might limit southward expansion of this species [59]. At the same time, six more indigenous mosquito species, Culex theileri [60], Anopheles maculipennis sensu lato, Coquillettidia richiardii [61], Aedimorphus vexans, Dahliana geniculata [Aedes geniculatus] and Ochlerotatus caspius [62][63][64] have recently been found infected by D. immitis in nature. All of the potential indigenous vectors are highly mammophylic and anthropophylic (excluding some members of An. maculipennis complex and Cx. theileri that only occasionally feed on humans) and could increase transmission rate of these filarioids.
Phortica variegata, the vector of T. callipaeda, was studied in an area of Italy where dog thelaziosis is highly prevalent [16] and found to be more active at 20-25°C and 50-75% RH during July-August in southern Italy [19]. Suitable environments for the geographic distribution and development of P. variegata across Italy and Europe were predicted using a desktop implementation of the Genetic Algorithm for Rule-Set Prediction and all recent reports of T. callipaeda fall within the suitable areas indicated by the model [12,14,15,65]. Based on this model, the number of reports of T. callipaeda infestation may be expected to increase over the next years in areas where it is now considered as non-endemic.

Host-vector interactions
The availability of suitable hosts for a given VBH and the vector's feeding behaviour are among the most important factors impacting on VBH distribution. For example, domestic dogs are excellent reservoirs of filarioids, being able to survive for a long time with a considerable worm burden, to harbour different species of filarioids at the same time and to provide infectious microfilariae for competent vectors all over their season of activity. Indeed, among filarioids only D. immitis can cause a fatal and severe disease, but presently the majority of dogs harbour a low-medium burden of nematodes, and they show a few symptoms with chronic progression in almost all the cases [66]. Without a doubt, in order to act as good reservoirs of VBH dogs also need to be attractive for competent mosquito vectors as well as tolerant to mosquito bites. For example, in a heartworm endemic north-eastern area of Italy, 70% of Cx. p. pipiens and 90% of Oc. caspius collected using dog-baited traps from June to September were engorged [67] and it was shown that the number of bites/dog/night can vary according to the vector density, weather conditions and dog size (average of 32.4 and maximum of 81 bites/dog/night). However, the attractiveness and tolerance to mosquitoes of other mammalian species should be considered when studying the potential reservoirs for filarioids in nature. Indeed, dogs are significantly more attractive to eight species of mosquitos (Aedimorphus taeniorhynchus [Aedes taeniorhynchus], Culex pipiens quinquefasciatus, An. maculipennis, Oc. caspius, Culiseta annulata, Ochlerotatus scapularis, Culex declarator and Cx. p. pipiens) than cats [68,69]. In spite of this, cats are not good reservoirs for D. immitis, mainly due to host resistance, as inferred by the low adult worm burden in natural and experimental infections, the long prepatent period (8 months), the low level and short duration of microfilaraemia and life span of adult worms (2-3 years) in this host species [70]. Interestingly, some species or strains of mosquitoes displayed inherent mechanisms of defence (refractoriness) to infestation such as the ability of the cibarial armature to destroy microfilariae, the anticoagulant activity of mosquito salivary proteins on the bolus containing microfilariae and other arthropod immunological responses to larvae [71].
Prevalence of microfilaraemic dogs and presence and abundance of competent vectors also affect the rate of infestation within a given mosquito population, which, in turn, is directly related to the risk for a native dog to be infested. In an endemic area of north-eastern Italy out of 40,000 culicids captured from May to October, and screened for D. immitis and D. repens with a realtime polymerase chain reaction (PCR), Cx. p. pipiens, Oc. caspius and Am. vexans were found positive for D. immitis with an estimated rate of infestation ranging from 0.21 to 1.11%, according to date and site [64]. In the same study, D. repens was found in Cx. p. pipiens only (rate of infestation of 0.23-0.71%). Interestingly, the rate of infestations did not vary significantly according to season, indicating that in spring a certain number of dogs may be ready to act as reservoirs and thus needing prolonged preventive treatments. In Turkey, an infestation rate of 0.41% and of 0.12% was recorded in Am. vexans, the main vector of D. immitis in this area, and in Cx. p. pipiens, respectively [63]. By combining the rate of infestations with the bites/dog/night numbers above for Cx. p. pipiens in north-eastern Italy, it can be speculated that a dog living in an endemic area has a chance to encounter an infected mosquito every 6 nights during the low abundance mosquito period and every 1.2 nights during the high abundance period of the summer. For sure, this calculation does not take into account the presence and the abundance of St. albopicta, which is now established in many areas of southern Europe throughout the year [72]. Stegomyia albopicta, a vector of D. immitis [46,73] and of D. repens [74] is active throughout the whole day and year in southern areas, especially in urban habitats. This scenario might be further complicated in the future by the introduction of new invasive mosquito species, such as Hulecoeteomyia koreica [Aedes koreicus], which is a potential vector of D. immitis in Belgium [75] and north-eastern Italy [76]. This species is colonizing colder environments, therefore increasing the possibility to enlarge the area at risk for dirofilariosis in Europe. The factors enhancing the exposure of the host to the vector (i.e., the dog's size, the age and the outside habitation) may further increase the risk of D. immitis infestation [66,77]. Other variables that are reported as risk factors, such as the sex, the length of the coat and dog's activities (i.e., guard, hunting, stray dogs vs. pet dogs) are likely to be biased by confounding factors, such as male dogs that are used as guard dogs and kept outside day and night.
In Europe, cats were found infected by D. immitis mainly in Italy, France and Portugal [3]. In Italy, the prevalence of feline heartworm disease has been approximately estimated as high as 10% of the known prevalence of the infestation in dogs [70]. However, filarioid infestation in cats can occur also in low endemic areas, as reported in central Italy [78]. Due to the very low worm burden usually found in cats, these animals are regarded as "victims" rather than reservoir of Dirofilaria spp. On the contrary, red foxes (Vulpes vulpes) were found infected by D. immitis in Italy, Spain and Bulgaria [3], with prevalence up to 32% in irrigated areas of Spain [79]. In Italy, out of 132 red foxes examined, 25% harboured microfilariae of D. immitis, 0.7% of D. repens, 15% of A. reconditum and 2.3% of A. dracunculoides [80]. Other hosts found infected with D. immitis are wolves (Canis lupus), in Belarus, Italy and Spain [48,81], jackals (Canis aureus) in Bulgaria [82] and otters (Lutra lutra) in Portugal and Spain [83]. The first European record of D. immitis in ferrets (Mustela putorius putorius) has also been reported [84], with a particular and aberrant larval migration to the central nervous system. All these hosts are likely to represent an epi-phenomenon of dog infection, with the exception of red foxes, which may act as a wild reservoir of the infection.
In the case of T. callipaeda, the reasons underlying the steady spread of this nematode throughout many European countries are not clearly understood, but it seems that the same zoonotic strain of T. callipaeda circulates in the continent within different animal species and humans [18]. The occurrence of very high prevalence of thelaziosis by T. callipaeda in foxes (49.3%) as well as in other wild carnivore species (i.e., wolves, beech martens, brown hares, and wild cats) in some areas of southern Italy where canine thelaziosis is highly prevalent (i.e., about 60% of dogs) indicates the status of hyper-endemicity of eyeworm infestation in this area and the primary role foxes play as reservoirs of the infestation [10]. The seasonality and crepuscular activity of P. variegata nicely overlaps the behaviour of those wild species hosts. The aforementioned ecological considerations are supported by molecular data on the occurrence of a single haplotype (i.e., h1) of T. callipaeda among different host species in the study area [18]. The same h1 was found in other European countries, irrespective of the host species from which they were collected [18]. These molecular findings and studies on P. variegata indicate a high level of affinity of the nematode for its vector [18,19,85] and low degree of specificity for definitive hosts. These results support the existence of a sylvatic life cycle for T. callipaeda and indicate that the infestation is mainly maintained by a large number of wildlife species that, altogether, could play a role in spreading the disease in many previously non endemic areas of Europe [11,[13][14][15][16]86]. Finally, the high prevalence of eyeworms in dogs and wildlife should represent an alert for human populations considering the difficulties in the differential diagnosis of the infection.

Impact of VBH on humans
Among the zoonotic filarioids, D. immitis and D. repens probably represent the species more frequently reported in humans where they are detected predominantly in the subcutaneous tissues, pulmonary vessels, testicles and also in the central nervous system, causing a range of clinical manifestations from asymptomatic to, more rarely, fatal syndromes [1,2,66]. In addition, human infestations by Dirofilaria spp. often induce nodular lesions, which may be erroneously diagnosed as cancers, hence representing a further challenge to physicians [2]. While D. immitis is the main agent of human dirofilariosis in the Americas [28,66], D. repens has been accounted for a long time as the sole species that infest humans in Europe [87,88]. For example, 28 cases of human dirofilariosis from the Old World erroneously attributed to D. immitis were reviewed and re-attributed to D. repens [88]. However, cases of human dirofilariosis by D. immitis have been recently described in Italy, Greece and Spain [89][90][91] and this trend is at an increase in Europe, most likely paralleling the spread of infestation in dogs in central and north-eastern countries (e.g., south of Switzerland, Czech Republic, Hungary, Serbia and Slovak Republic) [5,7,[48][49][50][51][52]. Hundreds of cases of human infestation by VBH have been reported worldwide [1], new cases continue to be reported from new geographic areas and it is likely that many more cases occur and are either unrecognized or go unreported. This is the case of O. lupi, which has probably been misdiagnosed for a long time with other filarioids localizing in the eyes. Indeed, O. lupi has only been suspected to act as a causative agent of infestation in humans until recently [92], when this species has been unambiguously identified morphologically and molecularly in two patients from Turkey and one from Tunisia who exhibited clinical features similar to those of the infestation in dogs [92,93]. Human thelaziosis is a condition described in several areas of the former Soviet Union and Asian continent (e.g., China, Korea, Japan, Indonesia, Thailand, Taiwan and India), predominantly in poor, rural communities with low health and socio-economic standards, particularly where domestic dogs and other animals (e.g., cats and foxes) are heavily affected and live in close contact with humans [94]. The first four cases of human thelaziosis in Europe were diagnosed in patients coming from the north-west of Italy, south-eastern France [20] and Spain [21], where the infestation had been previously reported in dogs, cats and foxes [11,14,16]. The clinical presentation is characterized by mild conjunctivitis, follicular hypertrophy of the conjunctiva, foreign body sensation, epiphora, itchiness, congestion, swelling, hypersensitivity to light, and keratitis.
Considering the lack of awareness for physicians concerning such exotic parasites (e.g., O. lupi) and the possibility of misidentifications, the impact of VHD on human populations in Europe, mainly in remote rural areas, is much likely underestimated at present.

Managing VBH in dogs and humans Prevention
From the picture above it emerges how difficult the prevention and the treatment of VBH in endemic areas may be. This is mostly an issue for dirofilarioses in dogs which, in turn, can be easily prevented with a number of macrocyclic lactones administered in a way to kill D. immitis or D. repens larvae before they develop into adults in the heart/lungs or the subcutis, respectively. Several molecules are available in chewable tablets, spot on and injectable formulations administered with different protocols (Table 1).
Ivermectin is licensed in the Europe to prevent infestations by D. immitis and D. repens, while spot-on formulations containing moxidectin and selamectin, the oral products containing milbemycin oxime may be further suitable choices for the prevention of D. immitis. The injectable long lasting formulation containing moxidectin showed to be effective in controlling D. immitis and D. repens infestations for a period of 6 months after a single administration [95,96]. The duration of monthly chemoprophylaxis against D. immitis (i.e., year round, six months, or only during the vector season) has been debated for long time [97][98][99]. Current guidelines on management of D. immitis infestation in dogs promoted by the European Scientific Counsel on Companion Animal Parasites (ESCCAP) and by the American Heartworm Society (AHS) suggest extending treatment to 7-8 months or even the year round. The rationale for that relies on the occurrence of certain mosquito vectors, such as St. albopicta, which may survive in temperate areas as adults even during winter and, at least, for nine months per year [100].
In addition, the use of broad-spectrum drug formulations enhance owner compliance and assist continued control of other helminths [99] and of certain ectoparasites according to the associated molecules (Table 1). Interestingly, the massive use of preventive measures against D. immitis infestation showed a decrease in the prevalence of infestation of unprotected dogs living in the same area, through the reduction of reservoir host population. This is the case of some areas of northern Italy where D. immitis was regarded as hyper-endemic until 20 years ago, whereas its prevalence decreased over the last decades [47]. Based on this evidence, preventative chemoprophylaxis should be effectively employed also in communities where heartworm prevalence is low or where it is considered emerging [101]. No data is available on the efficacy of macrocyclic lactones against minor species of filarioids (e.g., A. reconditum, A. dracunculoides and Cercopithifilaria spp.) infesting dogs. Consequently, their prevention currently mostly relies on the vector control [37]. Preventing the contact with the fly intermediate host of T. callipaeda by the use of bed nets has been recommended for avoiding human infestation [94]. No information is available on the usefulness of any drug as repellents on animals against P. variegata.

Treatments
The arsenical melarsomine dihydrochloride is the adulticide compound used for the treatment of canine heartworm, associated to confinement of dogs in cages during and for about a month after the treatment period, in order to prevent potentially fatal pulmonary thromboembolism after the death of the heartworms. Melarsomine is usually injected intramuscularly at the dose of 2.5 mg/kg either in a two-step (first intramuscular injection followed by the second 24 hr later) or threestep (first injection followed by the two-dose protocol 4-6 weeks later) regimen. Even though the three-dose scheme is indicated for those animals with a relevant risk of pulmonary thromboembolism post-treatment, this protocol is recommend by the AHS guidelines for the therapy of all infected dogs [101]. Ivermectin may kill adults of D. immitis if administered monthly at the preventive dosage of 6-12 μg/kg for not less than 16-30 months [102]. However, the AHS discourages the extra label use of macrocyclic lactones as primary adulticides albeit their partial efficacy against microfilariae [101]. In fact, such prolonged treatment period does not prevent from the onset of cardiopulmonary damage in the infected dogs, thus impairing a full clinical recovery [103,104]. The administration of a macrocyclic lactone for up to 6 months before injecting melarsomine can be beneficial in dogs not requiring urgent therapy, because it can reduce parasite burden and permits immature filarioids to reach adulthood at which time they are fully susceptible to adulticide [66,105]. A novel approach for the treatment of cardiopulmonary dirofilariosis is targeting the Wolbachia rickettsial endosymbionts. Treatment with tetracyclines has been reported to damage D. immitis, even causing death of adult worms [106]. Long-lasting administration of both doxycycline and ivermectin before or in the place of melarsomine injections can eliminate adult worms and also reduce risk of thromboembolism. Therefore, it has been suggested that a combination of doxycycline (10 mg/kg die for 30 days) and ivermectin (6 mcg/kg every 15 days for 6 months) has a potential efficacy, as high as 73%, in the adulticide therapy in dogs infested with D. immitis [107,108]. Although other filarioids of dogs, but not D. immitis, are considered less clinically relevant, microfilaricide treatment is required to control D. repens microfilariaassociated syndromes (e.g., cutaneous erythema and ulcerative pruritic lesions) and to decrease the risk of human and animal infestation in endemic areas. However, only little information is available for the treatment of subcutaneous filariosis by D. repens, such as a combination of injectable melarsomine and oral administration of macrocyclic lactones [109]. The use of prolonged selamectin or moxidectin administration in treating dogs infected by D. repens is reputed effective [110,111]. The latter molecule also showed a high degree of efficacy in treating D. repens infection, including potential ability to kill adults, after a single administration [112][113][114]. Surgical options usually rely on heartworm removal by the use of flexible alligator forceps with the aid of fluoroscopy-or trans-oesophageal echocardiography-guides, but the success of these procedures may be influenced by several factors [115].
Although minor species of filarioids infesting dogs, (e.g., A. reconditum, A. dracunculoides and Cercopithifilaria spp.), are considered clinically irrelevant, microfilaricide treatment should be always advocated to limit the reservoir function of infected hosts. Though reports on microfilaricide treatment for minor species are scant, evidence suggests that macrocyclic lactones (e.g., ivermectin, selamectin and moxidectin) are effective against patent infestation when administered at the same dosage recommended for D. immitis [116,117].
As far as O. lupi infestation, surgical removal of the nodules containing the worms remains the only curative treatment for ocular onchocercosis, even thought developmental stages present in periocular tissues and other parts of the body may cause relapses [32]. No specific pharmacological treatments have been reported yet for O. lupi infestation in dogs.
Treatment of domestic animals infested by T. callipaeda should be carried out and the topic instillation of organophosphates [118] or moxidectin 1% [119] showed to be highly effective. Imidacloprid 10% and moxidectin 2.5% in spot-on formulation was also effective for the control of dog thelaziosis within five (90.47%) to nine (95.23%) days after treatment [120], allowing to overcome problems due to the mechanical removal of parasites or to the restraining of the animals for the local instillation of drugs in the eyes. The administration of an injectable sustained-release formulation of moxidectin and of a monthly treatment with milbemycin oxime provided some seasonal protection against T. callipaeda infestation in dogs from an endemic area of northern Italy [121,122]. Such a chemoprophylaxis approach would more likely reduce the prevalence of dog thelaziosis, and therefore the risk for human infestations in endemic areas.

Diagnosis
Laboratory diagnosis of infestations caused by D. immitis, D. repens or A. reconditum is achieved through classical detection of circulating microfilariae, parasite antigens and/or by genetic tools. Microfilariae can be identified in the bloodstream of infected animals by microscopic techniques, using the Knott's test, which is the gold standard method [66]. Blood circulating microfilariae of D. immitis should be discriminated from those of other filarioids that do not infest the heart chambers and arteries (i.e. D. repens and A. reconditum). Key diagnostic features are differences in morphology and size measurements of particular structures. Head of D. immitis is slightly tapered, while that of A. reconditum and D. repens is blunt. The use of fixation in 2% formalin in the Knott's test can cause a distinctive artefact in the tail of A. reconditum and D. repens which become curved ("buttonhook" or "umbrella" tail), while that of D. immitis remains straight [123,124]. Given that the occurrence of such an artefact may vary considerably, the possibility that A. reconditum and D. repens larvae present a straight tail cannot be ruled out. Hence, the body length becomes discriminatory for the identification at the species level. Indeed, microfilariae of D. immitis are 260-340 mm in length and 5-7.5 mm in width, while those of D. repens are longer and slightly wider (325-380 × 5-8 mm) and those of A. reconditum are smaller and thinner (240-290 × 4.5-5.5 mm) [123,124]. However, microscopic detection of circulating microfilariae may lack in sensitivity. Single-sex infestations, low parasite burdens, immune reactions or past administration of parasiticides with microfilaricidal activity may cause lack of circulating larvae in up to 20-30% of dogs infected by D. immitis [66]. An alternative method for diagnosing D. immitis infestation in dogs is the use of commercial kits for the detection of antigens released in the blood by adult females. However, some microfilaraemic animals may score negative at these tests for the low worm burden or for the persistence of microfilariae after the death of adult worms [66,112,113]. No similar tests are available for the other filarioids. Other diagnostic tools may include echocardiography, which has high sensitivity although requiring high professional expertise while performing the test [125].
Dermal microfilariae of O. lupi and Cercopithifilaria spp. can be detected by skin biopsies. Interestingly, it has been demonstrated that microfilariae of the latter species are unevenly distributed on the body of an infected dog with, however, higher frequencies on the interscapular region and on the head, where the tick vectors usually attach [38]. It should also be taken into account that at least three Cercopithifilaria spp., two still unidentified at the species level, may infest dogs. However, dermal microfilariae of these three species can be differentiated morphologically based on their length and presence/absence of lateral alae [36].
Recent molecular-based assays have been reported for the unequivocal identification of filarioids, irrespective of their life cycle stage. Ribosomal or mitochondrial DNA sequence fragments of D. immitis, D. repens and A. reconditum may be amplified and analysed with a restriction fragment length polymorphism, specific PCR amplifications or with primers yielding species-specific amplicons [126,127] and their usefulness has been demonstrated in epidemiological and clinical studies [112,128,129]. Recently, a duplex real-time PCR has been assessed for the discrimination between D. immitis and D. repens and their quantification in blood samples and mosquitoes [130]. A multiplex PCR based on the amplification of a mitochondrial gene of bloodcirculating microfilariae of D. immitis, A. reconditum and D. repens and of Cercopithifilaria spp. has also been shown to be useful for their molecular detection and differentiation in blood and skin samples [131]. Finally, a multicentre study in the Mediterranean area proved that the three species of Cercopithifilaria affecting dogs might be discriminated from each other by differences in mitochondrial cox1 and ribosomal 12S sequences [36].

Conclusions
Although great scientific achievements have been gained over the past decades on several aspects of the biology, epidemiology, control and treatment for many VBH (e.g., D. immitis and D. repens), most of them have only been recently known to science, thus they remain enigmatic in many ways. The increasing trend of VBH in Europe is most likely due to the spreading process of several arthropod vector species and highlights the need for actions focussing on control of the vectors in the environment and the protection of animals at individual and population levels. Indeed public health authorities should be concerned about the potential risk of introduction and establishment of new and exotic vectors, which may alter the VBH scenario in a manner that may not be easily foreseeable.
The biological mechanisms behind the increased number of cases of VBH in Europe remain uncertain and research on the role played by insect vectors for many of them is lacking. This is only partially due to the complexity of the relationship between pathogen, host and vector. Indeed, some VBH are relatively poorly investigated and this lack of awareness represents a major constraint to their successful management and control in endemic and non-endemic areas. For example, veterinary/medical surveillance accompanied by entomological surveillance is essential to prevent the spread of filaroids and to evaluate the risk of zoonotic filariosis outbreaks. There is a need to monitor closely the changing epidemiology of VBH in order to predict their future distribution, particularly in light of their constant spread and of socio-economic and political events. Under the above circumstances, the economic crisis and the subsequent population movements from southern to central and northern European areas may render it difficult to afford effective therapeutic treatments and a correct management of the environment, towards the reduction of arthropod vector breeding sites.
In addition, although basic and applied research in the biology of insect vectors is often considered the 'Cinderella' in the political agenda of governmental funding agencies, these are essential for controlling arthropods and VBH, especially given the introduction of European directives which limit the number of available biocides (e.g. 98/8/EC) and ban the aerial use of insecticides (e.g., P6-TA-(2009) 0010) within the European Union. Since the range of alternatives is limited, identifying novel control strategies is essential.