s2fieldsofstudy: Biology Medicine

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Increased expression of claudin-17 promotes a malignant phenotype in hepatocyte via Tyk2/Stat3 signaling and is associated with poor prognosis in patients with hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in Asia; however, the molecular mechanism in its tumorigenesis remains unclear. Abnormal expression of claudins (CLDNs), a family of tight junction (TJ) proteins, plays an important role in the metastatic phenotype of epithelial-derived tumors by affecting tight junction structure, function and related cellular signaling pathways. In a previous study, we used a tissue chip assay to identify CLDN17 as an upregulated gene in HCC. Here we aimed to use molecular biology technology to explore the effect of CLDN17 on the malignant phenotype of HCC and the underlying molecular mechanism, with the objective of identifying a new target for HCC treatment and the control of HCC metastasis. Method The expression levels of CLDN17 in HCC tissues and histologically non-neoplastic hepatic tissues were explored by immunohistochemistry. Stable transfection of the hepatocyte line HL7702 with CLDN17 was detected by real-time polymerase chain reaction (PCR), western blotting and immunofluorescence. The impact of CLDN17 on the malignant phenotype of HL7702 cells in vitro was assessed by a Cell Counting Kit-8 (CCK8) assay, a Transwell assay and a wound-healing experiment. Western blotting was utilized to detect the activation state of Tyrosine kinase 2 (Tyk2) / signal transducer and activator of transcription3 (Stat3) pathway. A Tyk2 RNA interference (RNAi) was utilized to determine the impact of the Tyk2/Stat3 signaling pathway on the malignant phenotype of hepatocytes. Results In this work, our research group first found that CLDN17 was highly expressed in HCC tissues and was associated with poor prognosis. In addition, we demonstrated that CLDN17 affected the Stat3 signaling pathway via Tyk2 and ultimately enhanced the migration ability of hepatocytes. Conclusion In conclusion, we confirmed that the upregulated expression of CLDN17 significantly enhances the migration ability of hepatocytes in vitro and we found that the activation of the Stat3 pathway by Tyk2 may an important mechanism by which CLDN17 promotes aggressiveness in hepatocytes.

Background
Previous studies have shown that the metastasis of epithelial-derived tumors is accompanied by abnormalities in tight junction (TJ) structure and function [1,2]. Claudins (CLDNs) are the key proteins that form TJ, and accumulating evidence suggests that tumor cells frequently exhibit changes in the expression and localization of CLDNs [3]. For example, CLDN1 demonstrated to be overexpressed in colorectal cancer (CRC) compared with the level in the normal mucosa, and CLDN1 targeting with an anti-CLDN1 monoclonal antibody (mAb) resulted in decreased growth and survival of colorectal cancer (CRC) cells, suggesting that CLDN1 could be a new potential therapeutic target for CRC [4]. In addition, a database-augmented, exosome-based mass spectrometry approach identified circulating CLDN3 as a biomarker in patients with prostate cancer [5]. Moreover, high-level cytoplasmic CLDN3 expression is an independent predictor of poor survival in patients with breast cancer [6]. The TJ protein CLDN4 has been reported to be overexpressed in advanced ovarian cancer (OC) and Kaplan-Meier survival analyses and the log-rank test suggest that high expression of CLDN4 may have prognostic value in OC [7]. These observations revealed that the alterations in CLDNs expression may be related to tumorigenesis and cancer progression in various types of human carcinoma.
Additionally, CLDNs have been shown to participate in the transduction of intracellular/extracellular signals and may be related to tumorigenesis and cancer progression in human various carcinomas [8,9]. For instance, genetic and pharmacological studies confirmed that the expression of CLDN3 was downregulated in colon cancer and that the loss of CLDN3 induced Wnt/β-catenin activation in a transducer and activator of transcription 3 (Stat3)-dependent manner to promote colon cancer malignancy [10]. Besides, a recent study revealed that enhanced CLDN18 expression activated ERK1/2 to contributed to the malignant potentials of bile duct cancer [11]. Our preliminary work showed that CLDN17 was strongly expressed in HCC tissues and cell lines and weakly expressed in non-neoplastic tissues and hepatocyte lines, which revealed that upregulated CLDN17 expression may play a role in the development of HCC. Furthermore, gene chip screening revealed that CLDN17 overexpression activated the tyrosine kinase 2 (Tyk2)/Stat3 pathway signaling pathway. To date, there has been no report on the impact of CLDN17 on the malignant phenotype of hepatocytes. In this study, we utilized molecular biology and other techniques to study the role and mechanisms of CLDN17 in malignant phenotype of hepatocytes and to identify novel targets for HCC treatment and the control of early metastasis.

Cell culture
Human hepatocyte line (HL7702) and HCC cell lines (HepG2, Hep3B and Huh1) utilized in this study were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences. These cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator containing 5% CO 2 .

Plasmid construction and transfection
The plasmid p-EGFP-C1/CLDN17 (NM_012131) was constructed and amplified by KeyGen BioTech Company. Two micrograms of plasmid DNA was transfected into cells using the SuperFect Transfection Reagent (TaKaRa, Japan) according to the manufacturer's protocol. A cell line stably expressing CLDN17 was selected in medium containing G418 (Thermo Fisher Scientific, Waltham, MA).

Real-time polymerase chain reaction (PCR)
Total RNA was extracted using a Perfect Pure RNA Cultured Cell Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. Real-time PCR reactions was carried out as previously described [12]. The primer pairs used for CLDN17 and glyceraldehyde phosphate dehydrogenase (GAPDH) were as follows: CLDN17 forward (5′-ACCCAGCCATCCACATAG-3′) and reverse (5′-CCCTTGCTTCTTTCTGTTG-3′); and GAPDH forward (5'-AACGTGTCAGTCGTGGACCTG-3′) and reverse (5'-AGTGGGTGTCGCTGTFGAAGT-3′). The relative expression was based on the expression ratio of a target gene compared with that of GAPDH.

Western blotting
A bicinchoninic acid (BCA) Protein Assay Kit (Pierce Chemical Co., Rockford, Illinois, USA) was utilized to detect protein concentrations. Total protein (30 micrograms) was separated via 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto a nitrocellulose membrane (Millipore, Temecula, California, USA). Next, the membrane was blocked and investigated with the following primary antibodies: rabbit anti-human phospho-Stat1, rabbit anti-human Stat1, rabbit anti-human phospho-Stat3, rabbit anti-human Stat3, rabbit anti-human phospho-Tyk2, rabbit anti-human Tyk2, rabbit anti-human CLDN17 and mouse anti-human β-actin. After 3 washes with phosphate-buffered saline (PBS), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnologies, California, USA) at a 1:1000 dilution at 4°C. Immunoreactive bands were detected using ECL western blot reagents (GE, Fairfield, Connecticut, USA) and analyzed with Image Lab 6.0.1 Software from Bio-Rad Laboratories.

Immunofluorescence method
The cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, cat. no. 9002-93-1). Then, after blocking with 2% bovine serum albumin (Bote Biotechnological Corporation, Jilin, China) diluted in PBS for 30 min, the cells were probed with a primary rabbit anti-human CLDN17 antibody, which was diluted in blocking solution (1:1000 dilution) for 30 min at RT. The cells were incubated with Alexa Fluor®647-conjugated anti-rabbit IgG antibody (ab150093, Santa Cruz Biotechnologies, California, USA) at a 1:1000 dilution.

Cell counting Kit-8 assay
Cell proliferation curve generated by the colorimetric water-soluble tetrazolium salt assay using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) as the protocol. The cells were seeded into 96-well plates in triplicate, and cell proliferation was recorded per 12 h for 4 days.

Wound-healing assay
The cells were maintained in a monolayer at 70% confluence on 24-well plastic dishes and the monolayer was scratched with a 100-μl pipette tip. The wounds were photographed light microscope (E100, Nikon Instruments Inc., Japan) (magnification × 200) at the same location at 0, 12 and 24 h.

Transwell chamber method
The cells were grown in a monolayer at 90% convergence and were maintained in FBS-free medium for 12 h. Matrigel (BD Biosciences, cat. no. 356234) was added to the upper Boyden chamber (Millipore, Bedford, MA) in 24-well plates and the plates were maintained in a cell incubator at 37°C for 15 min. Then, medium containing chemotactic factors, which had been collected from the cell culture, was added to the 24-well plate. The cells were supplemented with Matrigel and cultured in a cell incubator at 37°C for 6 h.

RNA interference (RNAi) method
Frozen glycerol bacterial stocks containing pGCSILscramble and pGCSIL-Tyk2-RNAi were purchased from Nanjing KeyGen Biotech Co., Ltd. The target was Tyk2-RNAi (29473), and the control insert sequence was pGCSIL-scramble. HEK 293 T cells (0.2 × 107) were seeded and maintained for 24 h to achieve 70-80% confluence in 6-well dishes (Costar, Cam-bridge, MA). Three plasmids, including of pGCSIL-Tyk2-RNAi or pGCSIL-scramble, 5 μg of the packaging vector pHelper 1.0 and 5 μg of a vesicular stomatitis virus glycoprotein (VSVG) expression plasmid vector, were added to Opti-MEM, with a final volume of 1.0 ml. Then, 50 μl of Lipofectamine was added to 950 μl of FBS-free medium. These two solutions were mixed and added to the cells. Lentiviral particles were harvested 48 h after transfection, and the viral titer was determined by counting green fluorescent protein (GFP)-expressing cells under a fluorescence microscopy (Nikon Diaphot 300®) with filters 96 h after transfection.

Patients and tissue samples
Biopsies were collected from 52 patients with pathologically confirmed the diagnoses of HCC who received treatment at The First Bethune Hospital of Jilin University between June 2007 and May 2012. The patients were carefully chosen based on the following criteria: no history of radiotherapy or chemotherapy and no prior malignant disease. The grade and classification of the HCC patients were based on the American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) staging system. Thirty cases of histologically non-neoplastic hepatic tissues and 10 cases of cirrhosis tissues were also obtained from hepatitis B virus infected patients who were treated at The First Bethune Hospital, Jilin University during the period between October 2006 and September 2011 that were identified to be histologically non-neoplastic. There were 16 men and 14 women with an average age of 49 years. The medical records of the patients were reviewed to determine the clinical and pathological characteristics.

Immunohistochemistry
An immunohistochemistry was utilized to explore the expression patterns of CLDN17 in 52 HCC tissues, 10 cirrhosis tissues and 30 non-neoplastic hepatic tissues. Of the 52 cases, 42 cases exhibited HBsAg infection, 17 cases exhibited occurrence and metastasis, and 34 cases were coupled with cirrhosis. The experimental method was described previously [13], and the antibody utilized was a rabbit anti-human CLDN17 antibody. The evaluation of protein expression levels was based on the percentage of positively stained tumor cells in combination with the staining intensity as previously described [14].

Follow-up
The patients with a pathologically confirmed diagnosis of HCC were followed-up for 60 months after diagnosis to assess occurrence and metastasis and to determine survival. The survival status of the patients was determined through a telephone interview or an outpatient visit before December 2017.

Statistical methods
All of the experiments were repeated 3 times, and all of the data are based on the mean ± SD of at least 3 experimental results. The experimental results were analyzed using Student's t-test, and the prognostic significance and value of CLDN17 expression was determined by the Chi-square test/Chi-square goodness-of-fit test. P < 0.05 was considered statistical significance.

Results
The expression of CLDN17 was upregulated in HCC cell lines and tissues Real-time PCR and western blotting were utilized to detect the expression of CLDN17 in the human hepatocyte line and the HCC cell lines (Huh1, HepG2 and Hep3B). We found that the mRNA and protein expression levels of CLDN17 were low or absent in the human hepatocyte line HL7702 but high in HCC cell lines Huh1, HepG2 and Hep3B (Fig. 1a-c). The protein expression of CLDN17 in hepatocyte tissues, cirrhosis tissues, tissues adjacent to the tumors and HCC tissues. Note: * represents P < 0.05, ** represents P < 0.01, compared with the empty vector groups CLDN17 expression was explored in 52 HCC tissues, 10 histologically non-neoplastic cirrhosis tissues, 10 histologically non-neoplastic tissues adjacent to the tumor and 30 histologically non-neoplastic hepatic tissues. As shown in Fig. 1e, the expression of CLDN17 in hepatic tissues and HCC tissues is located mainly in the cytoplasm and membrane. High expression of CLDN17 was observed in 53.8% (28/52) of HCC tissues 30.0% (3/10) of non-neoplastic cirrhosis tissues, 40.0% (4/10) of histologically non-neoplastic tissues adjacent to the tumor and in 40.0% (12/30) of hepatic tissues (P = 0.0001 < 0.001) ( Table 1).
The log-rank test was utilized to analyze correlations between CLDN17 and clinical survival. As shown in Fig.  1d, patients with positive expression of the CLDN17 protein in tumors (median survival, 46.15 months) had a notably shorter survival than those with negative expression of the CLDN17 protein (median survival, 57.79 months).
The relationships between CLDN17 and clinical pathological indicators were also analyzed, and the expression of CLDN17 was not associated with age (P = 1.000), HBsAg absent status (P = 1.000), cirrhosis (P = 1.000), serum AFP level (P = 0.637) of HCC patients or clinical staging (P = 1.000) of HCC patients.

Stable transfection of a hepatocyte line with CLDN17
The p-EGFP-C1/CLDN17 plasmid was utilized to transfect HL7702 cells. After G418 screening, a monoclonal strain of HL7702 cells was obtained, which was termed HL7702-CLDN17. Real-time PCR and western blotting were also utilized to detect the expression of CLDN17 in cultured cells. The results showed that the mRNA and protein expression levels of CLDN17 in the HL7702-CLDN17 group were notably higher than those in the empty vector groups (P = 0.0001 < 0.01; P = 0.0001 < 0.01, respectively) (Fig. 2a, b  and d). Immunofluorescence was utilized to detect the localization of CLDN17 in HL7702-CLDN17 cells. The results showed that the expression of CLDN17 was primarily localized to the cell cytoplasm and membrane (Fig. 2c). These results demonstrated that clonal HL7702 cell line that stably expressed CLDN17 had been successfully established.

The impact of CLDN17 on the proliferation and migration of hepatocyte lines
The growth curve for the HL7702 cell line was generated by the CCK-8 method. The data revealed that the proliferation rates of HL7702-CLDN17 cells at 48 and 72 h were notably higher than those of the empty vector groups (Fig. 3a). A wound-healing experiment was utilized to detect the impact of CLDN17 on the migration ability of hepatocytes (Fig.  3b). The results showed that the migration distances of HL7702-CLDN17 cells were substantially greater than those of the empty vector groups at 12 and 24 h (P = 0.0022, < 0.01). The Transwell chamber method was also utilized to detect the migration ability of hepatocytes. Twelve hours after the cells were seeded, the cells that invaded through the membrane of the chamber were observed. The results showed that the number of invasive cells in the HL7702-CLDN17 group was notably higher than in the empty vector groups (Fig. 3c and d). These results suggested that CLDN17 clearly promoted the proliferation and migration ability of hepatocytes in vitro. The impact of CLDN17 on the Tyk2/Stat3 signaling pathway in hepatocytes Western blotting was utilized to explore the activation state of the Tyk2/Stat3 pathway. The results showed that after the overexpression of CLDN17, the phosphorylation levels of Stat1, Stat3 and Tyk2 were substantially increased in the HL7702 cells ( Fig. 4a and b). These data suggested that CLDN17 upregulation notably enhanced the activation of the Tyk2/Stat3 signaling pathway in the HL7702 cell line.

The impact of Tyk2/Stat3 signaling pathway activation on the invasion and migration ability of hepatocytes
To determine the impact of the Tyk2/Stat3 signaling pathway on the invasion and migration ability of hepatocytes, the pGCSIL-scramble plasmid and the pGCSIL-Tyk2-RNAi plasmid were utilized to transfect HL7702-CLDN17 cells.
Western blotting was utilized to analyze Tyk2 expression and the phosphorylation level of Stat1 and Stat3 in these cells, and the results showed that the protein expression of Tyk2 was markedly downregulated in Tyk2-RNAi cells compared with the scramble group ( Fig. 5a and b). The data showed that after the knockdown of Tyk2, the phosphorylation level of Stat3 was notably downregulated while there was no significant change in the phosphorylation level of Stat1 in the HL7702 cell line that overexpressed CLDN17 (Fig. 5a). A Transwell chamber assay and wound-healing assay were utilized to analyze the effect of Tyk2 on the invasive and migration ability of the examined cells. The results showed that the number of invasive HL7702 cells in the CLDN17-expressing cells was notably decreased following Tyk2 silencing in CLDN17-expressing cells ( Fig. 5c and d). The migration distances of the Tyk2-RNAi cells were substantially shorter than those of the scramble group at 12 and 24 h (Fig. 5e).

Discussion
A number of studies have focused on the role of CLDNs in the tumorigenesis of HCC. For instance, CLDN1 has been described as a key factor in the entry of hepatitis C virus (HCV) into hepatocytes, and upregulated expression of CLDN1 was revealed to contribute to the promotion of epithelial mesenchymal transition (EMT) via the The corresponding statistical analysis of protein expression in the HL7702 cell line. Note: * represents P < 0.05, and ** represents P < 0.01 compared with the empty vector groups c-Abl/Raf/Ras/ERK signaling pathway [15,16]. Furthermore, it was demonstrated that CLDN3 is an epigenetically silenced tumor suppressor gene in HCC, and its overexpression notably inhibits metastasis by suppressing the EMT via the Wnt/β-catenin signaling pathway in HCC cells [13]. Besides, CLDN14 was epigenetically silenced via the trimethylation of lysine 27 on histone H3 (H3K27ME3) and was a novel prognostic biomarker in HCC [17]. Given the correlation between the expression levels of these CLDNs and the tumorigenesis of HCC, CLDNs represent potential novel therapeutic targets in patients with HCC. Fig. 3 The impact of CLDN17 on the proliferation and migration ability of cells in vitro. a A growth curve for the HL7702 cell line was generated by the CCK-8 method; (b) A wound healing assay was utilized to detect the migration ability of the HL7702 cell line in vitro; (c) The Transwell chamber method was utilized to detect the invasive ability of the HL7702 cell line in vitro; (d) The corresponding statistical analysis of invaded cells. Note: * represents P < 0.05 and ** represents P < 0.01 compared with the empty vector groups Fig. 4 The impact of CLDN17 on the Tyk2/Stat3 signaling pathway. a Western blotting was utilized to detect the activation of the Stat3 signaling pathway in the HL7702 cell line; (b) The corresponding statistical analysis of the activation status of various Stat3 pathway components. Note: * represents P < 0.05 and ** represents P < 0.01 compared with the empty vector groups CLDN17 is one of 27 members of the CLDN protein family, and our current understanding of the biological functions of CLDN17 is primarily limited to epithelial and epidermal permeability, barrier protection, and cell connections; reports on the relationship between CLDN17 and tumors are rare [18]. Our research group first found that CLDN17 expression was highly expressed in HCC tissues, and we speculated that the high expression of this gene may be involved with the tumorigenesis and progression in patients with HCC. Moreover, in the present study, we confirmed that CLDN17 markedly promotes the invasive ability of the hepatocyte line HL7702. Similar to our study, several studies have identified specific CLDNs as pro-oncogenes in human various cancers. For instance, previous work has shown that CLDN1 plays a key role in inflammation-induced growth and progression in patients with colorectal carcinoma [19]. Furthermore, Philip, R. et al. reported that CLDN7 expression in colorectal cancer contributes to motility and invasion by promoting a shift towards EMT by recruiting EpCAM towards TACE/ presenilin2 [20]. It was also revealed that CLDN7 is frequently overexpressed and promotes invasion in ovarian cancer [21]. However, in contrast to our results, other studies have shown that some CLDNs could be identified as tumor suppressor gene [22,23]. For instance, the expression of CLDN1 was reduced in stage II and III rectal cancer and was established as a factor that correlates clearly with recurrence and poor prognosis [24]. In addition, the expression of CLDN6 was demonstrated to be silenced in cervical carcinoma tissues, and the restoration of CLDN6 expression suppressed cell proliferation and colony formation in cervical carcinoma cells in vitro, and tumor growth in vivo [25]. One potential reason for this difference is that the functions of CLDNs may be specific and dependent on different interacting molecules in different cells [26,27]. In this manner, specific CLDNs RNAi was utilized to silence Tyk2 expression in CLDN17-expressing cells. a Western blotting was utilized to examine the effects of silencing Tyk2 and activating the Stat3 signaling pathway in the HL7702 cell line; (b) The corresponding statistical analysis of the activation of the Stat3 signaling pathway; (c) The Transwell chamber method was utilized to detect the impact of Tyk2 silencing on the invasive ability of the cells in vitro; (d) The corresponding statistical analysis of invaded cells; (e) A wound healing assay was utilized to detect the migration ability of the HL7702 cell line in vitro. Note: * represents P < 0.05 and ** represents P < 0.01 compared with the scramble group may have specific impacts on the biological behavior of a given tumor [28][29][30].
At present, our results first indicated that the CLDN17 was overexpressed and highly associated with metastatic progression and prognosis in patients with HCC. Moreover, the overexpression of CLDN17 markedly promoted the invasion and migration abilities of the hepatocyte line HL7702. Furthermore, we also performed an initial exploration of the molecular mechanism associated with this effect, and we found that CLDN17 upregulation affected the Stat3 signaling pathway via Tyk2 and ultimately enhanced the migration ability of hepatocytes. Considering the limited therapeutic options for patients with HCC, the role of CLDN17 as a therapeutic target merits further exploration.

Conclusion
There have been few reports on the roles of CLDN17 in tumors, and many problems related to the specific molecular mechanisms must still be researched. In the present study, we confirmed that the overexpression of CLDN17 notably enhanced the malignant phenotype of the hepatocytes. In addition, the induction of Tyk2/Stat3 signaling may be one of the most important mechanisms by which CLDN17 promotes the migration ability in hepatocytes.


s2fieldsofstudy: Biology Chemistry

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Added Value of Microscale Raman Chemical Analysis in Mild Traumatic Brain Injury (TBI): A Comparison with Macroscale MRI

: Di ﬀ use axonal injury and microhemorrhages, common complications after mild traumatic brain injury (mTBI), can lead to neurodegeneration and disability and have negative socioeconomic consequences. Magnetic resonance imaging (MRI) is conventionally used to study brain injuries in vivo, but microscale damage common in mTBI is challenging to detect. Raman microscopy is an e ﬀ ective diagnostic tool to investigate cells and tissue in a label-free manner, but the scanning mode of Raman microscopy is typically used only in vitro. Here, we show that Raman microscopy complements in vivo MRI, providing the vital information on the structural and molecular changes caused by mTBI in rats. We demonstrate that a method based on Raman microscopy allows us to detect structural damage invisible by conventional MRI and spot molecular changes in protein/ lipid concentrations caused by mild TBI.

INTRODUCTION
Traumatic brain injury (TBI) is the pathological alteration in brain functions caused by the impact of an object or the rapid acceleration/deceleration of the brain. In recent years, it has been recognized as an important health issue with a negative socioeconomic impact. 1,2 There are several gradations of TBI, mild, moderate, and severe, based on the Glasgow Coma Scale and the severity of symptoms. 3 Mild TBI (mTBI) is diagnosed in 80% of head trauma patients in, for example, the United States. 4 It is a common trauma that can occur on numerous occasions: in military conflicts; as a result of sporting activities, particularly in sports such as American football or boxing; and as a result of day-to-day activities associated with possible injuries (bicycle, motorcycle, and car accidents). 5 With the advancement of electrical bicycles, the danger of such occasions is only increasing. It was found that at least 30% of mTBI patients were not able to achieve full neurologic recovery and had developed prolonged cognitive and behavioral changes. 6 Magnetic resonance imaging (MRI) is a technique widely used for in vivo brain imaging in clinics. 7 It allows for noninvasive visualization of gray matter and white matter and detection of the damage caused by TBI, such as white matter shearing, foci of axonal injury, and small subacute hemorrhages. 8 These brain lesions were observed with MRI in patients with moderate and severe TBI. 9 However, an mTBI often shows no abnormalities on conventional MRI as it is not sensitive enough to detect the microbleedings and diffuse/ traumatic axonal injuries, which are the common complications of mTBI. 10,11 We have identified another technique, Raman microscopy, which can be complementary to MRI on the microscale.
Raman microscopy can thus be a viable addition to neuroimaging tools because it allows nondestructive analysis and imaging of biological materials with spatial resolutions in the micrometer range. 12 It is a label-free, optical method based on the inelastic laser light scattering from molecular vibrations inherent to any material. It is widely used in monitoring the state of materials, 13 chemical analysis, 14 cell biology, 15−17 histological and cancer studies, 18 etc. It was also shown that Raman microscopy allows label-free detection of cancer cells in the brain. 19 Raman spectroscopy was also used for analysis of biochemical changes in peripheral nerves after an injury 20 and to track the molecular changes on the surface of the brain hemispheres after TBI, 21 where temporal changes in tissue biochemistry were monitored.
In this study, a subsequent application of MRI and Raman has been carried out for the detection of mTBI. To the best of our knowledge, this has not yet been studied in detail and no analysis of deep brain structures after mTBI with Raman has been done in rats. The second and a more specific aim of this work is to investigate whether any changes in the corpus callosum region can be observed after mTBI using rats as experimental models because significant changes in the volume and morphology of the corpus callosum were reported in humans after severe TBI. 22

RESULTS AND DISCUSSION
Although mTBI is a major cause of disability, little is known about its underlying causes and mechanisms. Therefore, animal models are essential to study the underlying biomechanical, cellular, and molecular aspects of TBI, which cannot be studied in a clinical environment. 23 In the conducted experiments, an mTBI was induced by the so-called Marmarou weight-drop model. The severity of the TBI can be controlled by adjusting the mass of the weight and the drop height. 23 A replica of the Marmarou device was built for this purpose ( Figure 1A). Figure 1A−D presents the overall schematics of experiments: first, (A) dropping the weight, then (B) MRI investigations, followed by (C) creating slices in a cryomicrotome (cryotome), and (D) scanning with a Raman microscope.
To analyze the impact of this injury, we have conducted MRI experiments ( Figure 1B), which allow us to analyze the effects of mTBI in vivo. There are two reasons why microscopy images have an added value to MRI. First, although MRI images allow for deep in-tissue imaging, its spatial resolution is not as high as that of microscopic imaging (100 vs 1 μm). Second, MRI misses the specificity to detect the underlying biological effects of mTBI, whereas microscopic imaging might provide a valuable and complementary addition in this case. It is for these two reasons that we have chosen microscopy to provide additional means to look for details of the impact. Microscopic analysis requires the brain to be cut in thin slices ( Figure 1C). Among different microscopy techniques, we have chosen Raman microscopy imaging ( Figure 1D) because it allows us to image molecules, materials, and tissue in a labelfree manner by looking at the scattering of an impinged laser light vibration. We note that using Raman microscopy visualization of anatomical features in the scanned area is also possible by constructing heat maps based on the intensity of Raman peaks of interest ( Figure S1).
Experiments have been conducted using both techniques on a similar area in the brain before and after TBI. The brain of the rat was scanned with conventional T2-weighted MRI before, 1 day after, and 1 week after TBI with the purpose of identifying and imaging the damaged areas. Subsequently, the brains were extracted and subjected to Raman histological analysis with the focus on the corpus callosum region.
The anatomical T2-weighted images of an animal are shown in Figure 2. Here, one can detect the baseline scan before and 1 week after impact. Among the cut slides, we have chosen those slices where the corpus callosum and the lobes of the hippocampus are clearly visible. Because the anatomical T2 images are very similar in all animals, Figure 2 is a representative of all animals from this experiment. No significant changes were observed with conventional MRI ( Figure 2) in images obtained 1 day and 1 week after the TBI compared to those of the control rats. This supports the evidence that the induced impact produces only a mild TBI.
Subsequently, Raman microscopy has been used on similar slices in an effort to search for complementarity of the in vivo MRI and a higher resolution microscopy imaging modality. In Raman microscopy/spectroscopy, a monochromatic laser light (we have used 785 nm laser because it generates minimum fluorescence effects, which are viewed as side effects) excites a sample, whereas the scattered light containing information about vibrational frequencies of atoms of materials is detected and analyzed. Assignment of the peaks allows us to identify molecules. It was found that the peak intensity in the 2940 cm −1 region of the Raman neuronal spectrum is proportional to the neurofilament protein concentration and that of the peak in the 2855 cm −1 region corresponds to the myelin lipid concentration. 19 This allows us to track, in a label-free manner, the axon proteins/myelin turnover during peripheral nerve tissue regeneration.
The hierarchical clustering analysis has allowed us to visualize and assign the data points to different tissues present in the scanned area ( Figure 3A). The subsequent hierarchical

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Article clustering analysis of data sets containing data points from the corpus callosum revealed a histologically abnormal area (15− 20 μm in size) in a brain slice obtained from a rat 1 week after the medium force TBI ( Figure 3B).
This area had two times higher Raman signal intensity compared to that of the signal from the surrounding tissues and contained no peaks observed in the surrounding brain tissue. A similar signal pattern was noticed in the molecular fingerprint from dried rat blood ( Figure S2), for which a detailed Raman analysis of blood is carried out. This suggests that the observed abnormal area may be the result of microhemorrhages common in mTBI. 11 The absence of such areas in other TBI samples can be explained by either a not sufficient number of brain slices analyzed in this experiment or the fact that microhemorrhages are present only in a limited number in mTBI.
Subsequently, the data points corresponding to the corpus callosum were compared between samples with the focus on the 2800−3000 cm −1 Raman spectral region. A comparison of spectra between the control rats and mTBI rats has revealed a significant decrease of the 2940−2855 cm −1 peak ratios after mTBI with p = 0.001299 for TBI-130 versus the control rat. This condition was induced by a weight drop from 1.30 m. Also, a decrease in peak ratios was observed for TBI-145 induced by a weight drop from 1.45 m versus the control rat with p = 0.00076 ( Figure 4). However, it is important to note that only one TBI-145 rat was investigated with Raman microscopy, as the second rat was found dead next day after the impact. Because of the ethical consideration, it was decided not to subject additional animals for the 1.45 m weight drop impact; therefore, it is not possible to conclude whether the observed changes in the Raman spectrum were the result of the weight drop from 1.45 m or can be attributed to individual differences between animals. A somewhat extended analysis is presented in Figure S3, which shows data of 2940−2855 cm −1 peak ratios for individual rats.
Whether the observed differences between control and TBI-130 animals are the results of the changes because of induced TBI in all axons residing in the corpus callosum area or only in a limited number of axons will be the subject of future investigation. It can be thus concluded that Raman microscopy allows us to detect the damaged areas in brain after TBI and track the molecular changes on the tissue level. The main challenge of the presented approach is the fact that to perform such a study in the deep lying regions, like corpus callosum, cutting brain to thin slices is required, which is possible using MRI. Using Raman microscopy, this is possible with a higher resolution and sensitivity but only in vitro, which is destructive

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Article for animals. On the other hand, we note that once slices are cut, subsequent in vitro studies of tissue or cells using Raman spectroscopy are nondestructive and complementary to MRI because of the sensitivity and resolution. Furthermore, any detected abnormal molecular fingerprints can be extracted from a slice, cell, tissue, etc. and subsequently subjected to proteomic analysis or genomic sequencing. In addition, yet another complementarity between these techniques is linked with contrast enhancement by gold nanoparticles, which was reported for MRI 24 and surface-enhanced Raman scattering 25,26 for Raman detection, in which the detection and analysis can also be done on particles. 27,28

CONCLUSIONS
An approach combining MRI and Raman imaging is shown to be a viable tool to study mTBI on macro-and microscales. Raman microscopy provides the means to study mTBI at the tissue level, being complementary to MRI, which allows us to carry out analyses on the whole brain level. The TBI caused by the impact of a 450 g weight drop from 1.30 and 1.45 m heights did not produce lesions detectible with the conventional MRI, measured 1 day and 1 week after the impact. This provides a strong evidence that the applied force did not cause a severe TBI in rats. Using Raman microscopy, however, it was possible to detect even mTBI. This is evidenced by analyzing the decrease in the ratio of axon proteins to myeloid lipids as detected in the corpus callosum region after mTBI. The hierarchical clustering analysis allowed us to discriminate between different anatomical regions of the brain, detecting structural and molecular abnormalities inside the corpus callosum region, which is presumably the result of a microbleeding caused by mTBI. , and 40−60% humidity). 4.1.1. Induction of Mild Traumatic Brain Injury. Adult female Wistar rats (n = 8, 265 ± 16 g) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Five rats received a mild traumatic brain injury with varying severity (one from 1.0 m (TBI-100), two from 1.30 m (TBI-130), and two from 1.45 m (TBI-145)), and three rats were used as the control group (sham) and did not receive any impact.
Mild traumatic brain injury was induced using the Marmarou weight-drop model. 29 Therefore, rats were anesthetized with a mixture of isoflurane and O 2 (5% induction and 2% maintenance) and injected with 0.05 mg/kg buprenorphine (Temgesic, Indivior) subcutaneously. After 30 min, the heads of the rats were shaved, 100 μL of 2% xylocaine (AstraZeneca) was locally injected in the scalp, and an incision was made to expose the skull. A metallic disc with a diameter of 10 mm and thickness of 3 mm, which serves as a helmet, was glued onto the skull 1/3 before and 2/3 behind bregma. Next, the rat was placed on the custom-made foam bed with a mattress of a certain spring constant (Type E, Foam to Size, Ashland, VA) and positioned directly under a Plexiglas tube with a 450 g brass weight. The weight diameter and length were 18 and 210 mm, respectively. The rat was briefly detached from anesthesia, and the weight was dropped from heights of 1, 1.30, and 1.45 m. Immediately after the impact, the rat with the bed was moved away from the tube to prevent a second impact and the rat was reattached to the anesthesia. To reduce the hemodynamic shock, 1 mL of physiological solution (0.9% NaCl) was injected through a catheter that is placed in the lateral tail vein.
Subsequently, the helmet was removed and the incision was stitched. Additionally, the rats were imaged with a CT scan (X-Cube, Molecubes, Ghent, Belgium) to rule out any skull fractures because this is a criterion for euthanasia. To minimize the dose, a general-purpose, low-dose, single-bed position scan was performed. One day post impact, the rats received an extra dose of 0.05 mg/kg buprenorphine.
4.1.2. In Vivo Longitudinal MRI. MRI data were acquired on a 7T MRI scanner (BioSpin Pharmascan 70/16, Bruker, Ettlingen, Germany) using a volume of rat brain/mouse wholebody RF coil. All rats were imaged at baseline, and all TBI rats were also imaged 1 day post injury. TBI-100, TBI-130, and the sham rats were also imaged 1 week post injury. During imaging, the animals were under 2% isoflurane anesthesia (5% for induction), whereas their body temperature was kept constant with a circulating warm-water-heated blanket and bubble wrap; the respiration rate was monitored with a pressure pad.
At each time point, a whole brain anatomical T2-weighted scan was acquired using a rapid acquisition with refocused echoes (RARE) sequence: TR = 5.5 s, TE = 37 ms, RARE factor = 8, FOV = 2.5 × 2.5 cm 2 , and in-plane resolution of 109 × 109 μm 2 with 600 μm thick slices. There were 45 slices scanned, whereas the acquisition time was 12 min.
4.1.3. Raman Histological Analysis. 4.1.3.1. Sample Preparation. One week later, rats were euthanized with 100 mg/kg sodium pentobarbital (20%) (Kela NV, Hoogstraten, Belgium) and brains were extracted from their skulls. This was immediately followed by snap freezing of the brains in 2methylbutaan (Reagent-Plus 99%, Sigma Aldrich), which was cooled with liquid nitrogen. Sections were made with slice thickness 20 μm with a cryomicrotome (CM3050 S, Leica Microsystems, Belgium). Tissue slices were put on quartz slides (Ted Pella, Inc.) and fixed for 10 min with 4% formaldehyde (4078-9001, Klinipath). Tissue slices were submerged in the Hank's balanced salt solution (14025050, ThermoFisher) to avoid tissue detachment from slides and scanned with a Raman microscope. Raman microscopy analysis was conducted on corpus callosum areas.
4.1.3.2. Data Acquisition with Raman Microscopy. The Raman microscope (Alpha300R+) was equipped with a 785 nm laser (Toptica, Munich, Germany) and a CCD camera (ANDOR iDus 401 BR-DD, Belfast, Great Britain) cooled to −72°C. The laser power was set to 225 mW, whereas a water immersion 20×/0.6 NA objective (Nikon) was used for scanning. Tissue was mapped with a lateral resolution of 10 μm/pixel and an integration time of 1.5 s per data point. A total of 2500 spectra per slice were recorded.
4.1.3.3. Data Processing. The resulting Raman spectral data sets were processed in R using the HyperSpec package. 30 Background subtraction and baseline correction were done by applying the asymmetric least squares function and normalization by applying the area-under-curve method using the Baseline 31 and MALDIquant 32 packages, respectively. Hierarchical clustering analysis with the Bray−Curtis dissimilarity

ACS Omega
Article matrix was used to select the data points corresponding to the corpus callosum region and detect the tissue structural and molecular abnormalities. For the peak ratio analysis, the average mean spectrum calculated per slice was used.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02404.
Rat blood analysis ( Figure S1); raw heat maps generated by Raman area scan ( Figure S2); change in the Raman scattering intensity peak ratio ( Figure S3) (PDF)


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine Chemistry

text:
Albumin Overload and PINK1/Parkin Signaling-Related Mitophagy in Renal Tubular Epithelial Cells

Background Albumin, as a major urinary protein component, is a risk factor for chronic kidney disease progression. Mitochondrial dysfunction is one of the main causes of albumin-induced proximal tubule cells injury. Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria. The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate PINK1/Parkin-mediated mitophagy in renal tubular epithelial cells (TECs). Material/Methods Immunofluorescence assay and Western blot assay were used to detect the effects of albumin overload on autophagy marker protein LC3. Transmission electron microscopy and Western blot assay were used to investigate the role of albumin in mitochondrial injury. Western blot assay and co-localization of acidic lysosomes and mitochondria assay were employed to detect the activation of mitophagy induced by albumin. Finally, we explored the role of PINK1/Parkin signaling in albumin-induced mitophagy by inhibiting mitophagy by knockdown of PARK2 (Parkin) level. Results Immunofluorescence and Western blot results showed that the expression level of LC3-II increased, and the maximum increase point was observed after 8 h of albumin treatment. Transmission electron microscopy results demonstrated that albumin overload-induced mitochondrial injury and quantity of autophagosomes increased. Additionally, expression of PINK1 and cytosolic cytochrome C increased and mitochondria cytochrome C decreased in the albumin group. The co-localization of acidic lysosomes and mitochondria demonstrated that the number of albumin overload-induced mitophagy-positive dots increased. The transient transfection of PARK2 siRNA result showed knockdown of the expression level of PARK2 can inhibit mitophagy induced by albumin. Conclusions In conclusion, our study suggests that mitochondrial dysfunction activates the PINK1/Parkin signaling and mitophagy in renal tubular epithelial cells under albumin overload condition.

Background
Proteinuria, a universal feature in many renal glomerular diseases, can promote glomerular sclerosis and interstitial fibrosis [1][2][3]. Therefore, urinary proteins should not only be regarded as indicating glomerular injury, but also as a nephrotoxic factor for renal tubular cells [4,5]. Continuous high levels of proteinuria are an independent cause of progressive renal damage and play an important part in the progression from chronic kidney disease (CKD) to end-stage renal disease (ESRD) [6].
Albumin, as the main component of the protein in nephrotic urine, is normally filtered in the glomeruli and reabsorbed by Megalin and Cubilin receptors, which are co-localized in the renal proximal tubule [7,8]. Previous studies have shown that excessive albumin can induce apoptosis of renal tubular epithelial cells (TECs) [9,10]. Further studies have shown that mitochondrial damage is an important element in albumin overload-induced tubular apoptosis [11,12].
Mitophagy is a highly protected mechanism of lysosomal degradation for removing impaired mitochondria and preserving mitochondria in the appropriate condition [13,14]. Recent studies suggested that the Pink1/Parkin-mediated mitophagy pathway is one of the best studied mechanisms for mitophagy in mammalian cells. Parkin is a cytosolic E3 ubiquitin ligase which is encoded by the PARK2 gene. Pink1 can promote Parkin E3 ligase activity and recruit Parkin to mitochondria, then it promotes mitophagy [13,14]. It is closely related to many diseases, such as neurodegenerative diseases, blood diseases, tumors, and other diseases [14,15]. Mitophagy is also associated with renal injury. Chuanyan Zhao et al. showed that Pink1/Parkinmediated mitophagy plays a protective role in cisplatin-induced renal tubular epithelial cell injury [16]. However, whether Pink1/Parkin-mediated mitophagy is involved in albumin overload-induced renal TEC injury has yet to be determined. Therefore, our study investigated the possible mechanism of mitophagy by observing the effects of albumin overload on autophagy, mitochondrial damage, and Pink1/Parkin-mediated mitophagy in renal TECs.

Reagents and antibodies
The human proximal tubular epithelial HK-2 cell line was supplied by the China Center for Type Culture Collection (Wuhan, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium/F12 (DMEM/F12) were obtained from Gibco (Grand Island, NY, USA). Human serum albumin (HAS/ALB) was acquired from Sigma-Aldrich (St. Louis, MO, USA). LysoTracker Red and MitoTracker Green were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The antibodies used are described in Table 1.

Cell culture
Human proximal tubular epithelial HK-2 cells were cultured using DMEM/F12 medium containing 10% fetal calf serum, penicillin, and streptomycin in a 5% CO 2 incubator at 37°C. The medium was changed to serum-free medium at 24 h before incubation with endotoxin-free human serum albumin at 80% confluence.

Western blot analysis
The total soluble proteins, cytosolic fraction, and mitochondrial fraction were subjected to Western blot analysis. Total proteins were extracted with a protein extraction kit (Promega, USA) in accordance with the instructions. Isolation of mitochondria from HK-2 cells was performed using the Mitochondria/Cytosol Fractionation Kit (Beyotime, China). The cytosolic and mitochondrial fraction proteins were collected according to the manufacturer's instruction. We used a BCA assay kit (Thermo, USA) to measure protein concentrations. We separated 20 μg Membranes were blocked on a shaker at room temperature with 5% non-fat milk for 2 h, followed by incubated with primary antibodies described in Table1 overnight in TBS-T at 4ºC. The membranes were washed 3 times with TBS-T for 5 min on the shaker, then incubated with secondary antibody (Table 1) on the shaker at room temperature for 1 h. Chemiluminescence was imaged in a Molecular Imager Chemi DocTM XRS + WB with Image LabTM software (Bio-Rad, CA, USA), and band intensity was measured by ImageJ software.

Detection of mitophagy
Mitophagy was determined by the co-localization of mitochondria with lysosome. The cells were co-incubated with red-fluorescing LysoTracker Red (1 μM, Molecular Probes, Beyotime, China) and green-fluorescing MitoTracker Green (200 nM, Molecular Probes, Beyotime, China) for 20 min. Images were acquired with a Zeiss AXIO Observer D1 (Carl Zeiss, Oberkochen, Germany). The number of MitoTracker-and LysoTracker-positive dots was determined by manual counting of fluorescent puncta in more than 60 cells for each group.
Immunofluorescence staining for microtubule-associated LC3 and DAPI After treatment with albumin, the HK-2 cells were observed by optical microscopy and photographed. The cells were fixed in 4% paraformaldehyde for 15 min and washed 3 times with phosphate-buffered saline (PBS) for 5 min each time. Then, we added 0.1% Triton X-100 in PBS for permeabilization for 10 min and rinsed 3 times with PBS for 5 min each time. Then, the samples were blocked with BSA for 30 min at room temperature. Next, the HK-2 cells were incubated in a wet box at 4°C overnight with the anti-light chain 3B (LC3B) antibody (1: 50, Cell Signaling Technology, USA). After being rinsed 3 times in PBS for 5 min each time, the cells were incubated with an appropriate secondary antibody for 1 h in the dark at room temperature. Lastly, 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, C1002) was used as a counterstain. Results were evaluated by use of an OLYMPUS microscope (cellSens system) and an OLYMPUS confocal microscope (FV 1200). LC3-II dots were counted in individual HK-2 cells and the average of dots in at least 30 cells is presented in the figures.

Electron microscopy (EM)
HK-2 cells were fixed in 2.5% PBS-buffered glutaraldehyde at 4°C for 1 h after washing 3 times in PBS. Cells were fixed in 2.5% PBS-buffered glutaraldehyde at 4°C for 1 h after washing 3 times in PBS. Postfixation was performed in 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4), and dehydrated with an ethanol gradient. Then, we embedded the fixed HK-2 in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. The sections were observed under a transmission electron microscope (Hitachi, Tokyo, Japan).

The small interfering RNA (siRNA) and transfection
Small interfering RNA (siRNA) targeting PARK2 was purchased from Shanghai Gene Pharma Co., Ltd. (Shanghai, China). The PARK2 siRNA was transfected into cells with 5 μl/well of LipofectamineTM3000 in a 6-well plate following the manufacturer's instructions. After a total of 48 h of transfection, the maximum inhibitory effect of PARK2 expression was observed at 24 h. Untransfected cells were considered as a blank control, and a scrambled sequence siRNA was considered as the negative control.

Statistical analysis
Statistical analyses were performed with SPSS 16.0 (SPSS Inc., USA). ImageJ software was used to analyze the immunostaining. The t test and one-way ANOVA were used for comparing with baseline values and the variance between groups, respectively. Quantitative data are expressed as mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.

Effects of albumin overload on LC3 expression in HK-2 cells
We investigated the effects of albumin on LC3 expression by immunofluorescence and Western blot analysis. Cells were incubated with albumin (8 mg/ml) for various durations. The concentration of 8 mg/ml albumin was selected based on our previous studies. The time-related increase of LC3-II biosynthesis was evident. As shown in Figure 1A and 1C, in the 0 h group, only a few LC3-II dots were detected by immunofluorescence staining. Exposure to albumin (8 mg/ml) for 4 h or 12 h, the number of LC3-II-positive puncta significantly increased in cells treated with albumin for 8 h to 24 h (P<0.05), and the peak of the increase was at 8 h post-treatment. Similar patterns of LC3-II biosynthesis were observed by Western blot assay ( Figure 1B, 1D). These results suggest that albumin overload can induce autophagy. The expression level of LC3-II was markedly increased by treatment with 8 mg/ml albumin for 8 h, which is the most suitable concentration for studying the autophagy of renal TECs.

Effects of albumin overload on morphology of mitochondria
We observed the morphology of mitochondria by optical microscopy after the albumin treatment. The intact structure of mitochondria suffered severe damage in the albumin group compared with the control group ( Figure 2). We observed mitochondrial swelling, vacuolization, crista disruption, and disappearance of normal morphology in the albumin group ( Figure 2B), and the autophagosomes containing damaged mitochondria were also clearly evident ( Figure 2B-2D). These results suggest that autophagy activation is related with mitochondrial injury induced by albumin overload in HK-2 cells.

Effects of albumin on mitochondrial injury and mitophagy in HK-2 cells
To analyze the effects of albumin on mitochondrial injury, we examined the expression level of cytochrome C. Under normal conditions, cytochrome C resides in the mitochondrial membrane. With mitochondrial injury, cytochrome C translocates from the mitochondrial membrane to the cytoplasm. As shown in Figure 3, the level of cytosolic cytochrome C was notably increased by treatment with albumin (P<0.05), while mitochondrial cytochrome C was observably decreased (P<0.05) ( Figure 3A, 3B). These results indicate that albumin overload induced mitochondrial dysfunction and led to cytochrome C release from mitochondria to cytosol.
Albumin resulted in mitochondrial damage, so we next explored whether mitophagy was activated by albumin in HK-2  cells. We detected the expression level of PINK1 by Western blot and the results showed the HK-2 cells treated with albumin the expression level of PINK1 was greatly increased when compared to control cells (P<0.05) ( Figure 3C).
To further analyze the effect of albumin overload on mitophagy, we examined the quantification for dots of mitophagy by use of MitoTracker and LysoTracker co-staining. We observed that lysosomes and mitochondria were separated in the control group cells; however, the co-localization of lysosomes and mitochondria formed yellow fluorescence in the albumin-treated group cells, demonstrating that mitophagy was activated. The quantitative analysis of mitophagy-positive dots (yellow fluorescent dots) showed there were significantly more dots in the albumin-treated group compared with the control group (P<0.05) ( Figure 3D). These observations indicate albumin can induce mitophagy.

Effects of PARK2 knockdown on the mitophagy induced by albumin in HK-2 cells
We further investigated whether the PINK1/Parkin pathway was activated in albumin-induced mitophagy. First, we transfected the small interference RNA (siRNA) into HK-2 cells to knock down the level of PARK2. As shown in Figure 4A, 4B, 4D, and 4E, at 24 h after transfection, its normal levels were successfully reduced in 3# Parkin siRNA compared with the control group, so we chose 3# PARK2 siRNA for the following experiments.
Western blot analysis showed that, when compared to the control group, in the HK-2 cells treated with albumin, the expression levels of PARK2 (Parkin) was increased conspicuously,  which occurred concurrently with the change of autophagy markers, increases of LC3-II, and decrease of p62 level ( Figure 4C, 4F). Our findings indicated that the PINK1/Parkin pathway was activated in mitophagy induced by albumin, and silencing PARK2 reduced albumin-induced increase in the expressions of PARK2 (Parkin) and LC3-II. Simultaneously, after PARK2 siRNA transfection, albumin-induced mitophagy was remarkably inhibited in HK-2 cells, as indicated by MitoTracker

Discussion
Albumin, as the majority component of urinary protein, can induce TECs injury such as apoptosis, necrosis, or transdifferentiation, and can activate autophagy in TECs [17,18]. Growing evidence suggests that mitochondria are targets of albumininduced renal TECs injury [11,19]. Mitophagy plays a pivotal role in elimination of damaged mitochondria, which is mainly a process of sequestrating autophagic mitochondria and delivering them to lysosomes for hydrolytic degradation [20]. In the present research, we studied the effects of albumin overload on autophagy, mitochondrial damage, and mitophagy in renal proximal tubular (HK-2) cells in vitro.
Albumin overload triggers autophagic response in cultured TECs. We investigated the effects of various durations of albumin treatment on autophagy in HK-2, showing that the level of LC3-II was not markedly increased in the 4 h group, but it was significantly increased in the 8-h, 12-h, and the 24-h groups, especially in the 8-h albumin-treated group. As in our previous study, we found that 8 mg/ml is the optimal concentration for studying the damage and autophagy of TECs [17,18]. In brief, the albumin overload model using 8 mg/ml for 8 h may be the best condition for researching autophagy in HK-2 cells.
The albumin overload model can induce mitochondrial damage. We observed the morphology of mitochondria before and after albumin treatment. In this study, we found the manifestation of mitochondrial damage after albumin treatment, such as swelling, vacuolization, crista disruption, and normal morphological disappearance. At the same time, we observed the formation of double-membrane autophagosomes that contain damaged mitochondria, which suggests that autophagy activation is related with mitochondrial injury induced by albumin overload. In the 1950s, autophagy was first detected by transmission electron microscopy (TEM). Even now, TEM is still one of the best techniques for assessing autophagy by providing direct evidence, but TEM is problematic in quantitative studies for cell numbers/sections [20]. Thus, we examined the expression level of mitophagy marker PINK1 and mitochondrial injury marker cytochrome C, and found that the level of PINK1 was increased and cytochrome C was released from mitochondria to cytosol in the albumin group. As previously shown, mitochondrial depolarization accompanies cytochrome C release [21]. These results indicate that albumin overload induced mitochondrial damaged and activation of mitophagy.
Dysfunctional mitochondria can trigger their degeneration by mitophagy [14]. Several distinct mechanisms for mitophagy have been described in some recent papers. However, in mammalian cells, the Pink1-Parkin signaling pathway in mitophagy is the most documented [13,16,22]. The level of Pink1 is normally undetectable in most cells because mitochondrial Pink1 is cleaved by PARL and then degraded by mitochondrial peptidases [20,23]. However, when mitochondria are depolarized, Pink1 is no longer cleaved and becomes stabilized on the outer mitochondrial membrane [24]. PARK2 encodes for the E3 ubiquitin ligase Parkin. Pink1 promotes Parkin-mediated mitophagy by recruiting Parkin on mitochondria and promoting Parkin E3 ligase activity [24]. Subsequently, Parkin mediates the formation of 2 distinct poly-ubiquitin chains, linked through Lys 63 and Lys 27. Then, the adaptor protein p62 binds ubiquitinated mitochondrial proteins and LC3 on autophagosomes, recruiting autophagic membranes for mitochondrial clearance [24].
In the present study, we found that the expression level of PINK1 in the albumin group was significantly increased, and our quantitative analysis showed increased co-localization between mitochondria and lysosomes in the albumin group. Furthermore, in we found that knockdown of the PARK2 inhibited albumin-induced mitophagy, and the levels of autophagy-and mitophagy-related molecules were changed, including downregulation of LC3 and PARK2(Parkin) and upregulation of p62. Those results indicate that albumin overload can induce mitophagy activation in HK-2 cells, and the mitophagy was associated with activation of the PINK1/Parkin signaling pathway. Mitophagy is an important protective mechanism that removes damaged mitochondria and preserves a healthy mitochondrial population. Impaired mitophagy in kidney cells can accelerate CKD progression, and has been implicated in several kidney disease models. Zhan et al. showed that upregulation of myo-inositol oxygenase (MIOX) reduces Pink1 expression and inhibits mitochondrial autophagy, which can contribute to the pathogenesis of diabetic nephropathy [25]. However, the specific mechanism by which the PINK1/Parkin pathway is involved in albumin-induced mitophagy is still unclear, and the role of mitophagy in renal tubular interstitial injury caused by urinary protein needs further study.

Conclusions
In summary, our results show that albumin overload-induced mitochondrial dysfunction can activate the PINK1/Parkinmediated mitophagy in renal tubular epithelial cells. Albumin overload-induced mitophagy was increased in TECs, which suggests that the autophagy activated by urinary proteins may be a selective mitochondrial autophagy, and is mediated by the PINK1/Parkin pathway. This study further verified our previous research findings that, under urinary protein overload condition, autophagy activation can add adaptive reaction through eliminating injured mitochondria (mitophagy) in TECs.


Impact of Matrix Metalloproteinase 9 on COPD Development in Polish Patients: Genetic Polymorphism, Protein Level, and Their Relationship with Lung Function

Chronic obstructive pulmonary disease (COPD) is characterized by a decline of lung function and symptoms such as chronic bronchitis and emphysema leading from lung tissue destruction. Increased activity of matrix metalloproteinases (MMPs) and an imbalance between MMPs and their tissue inhibitors (TIMPs) are considered as factors influencing the pathogenesis of COPD. We investigated the role of genetic polymorphism and expression level of MMP-9 and concentration of its complexes with TIMPs in the development of COPD among Polish patients. We analyzed SNP in the promoter region of MMP-9 gene (rs3918242) using PCR-RFLP method among 335 COPD patients and 309 healthy individuals. Additionally, 60 COPD patients and 61 controls were tested for copy number variants (CNV) of MMP-9 (by quantitative real-time PCR) and serum levels of MMP-9 and its complexes with TIMP1 and TIMP2 (using ELISA). All subjects were analyzed for lung function using spirometry (FEV1% and FEV1/FVC parameters). We observed that allele and genotype frequencies of the SNP rs3918242, as well as the number of gene copies, were similar in COPD patient and controls groups. Serum levels of MMP-9 and MMP-9/TIMP1 complex were significantly higher in COPD patients in comparison to controls groups, although independently of analyzed gene polymorphisms. Additionally, the significant inverse relationships between parameters of lung function (FEV1% and FEV1/FVC) and proteins level were found in ridge regression models, especially we found that FEV1% decreased when MMP-9 level increased in controls and patients with COPD group. In conclusion, we found that COPD patients were predisposed to produce more MMP-9 and MMP-9/TIMP1 complex than healthy individuals. This phenomenon is probably associated with the disease-related lung environment but not with genetic features of the MMP-9.

Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation through the respiratory tract that is not fully reversible. The disease is associated with lung inflammatory response to harmful gases and dust, including cigarette smoke [1]. Moreover, COPD patients are affected with a pulmonary emphysema leading from lung tissue destruction and the decline of lung function as a result [2,3]. Thus, it is generally believed that mechanism underlying the COPD pathogenesis is dysregulation of proteases activity, especially when imbalance between proteases and antiproteases develops because of either the higher activity of proteases or dysfunction of protease inhibitors [3][4][5].  N, numbers of individuals; COPD, chronic obstructive pulmonary disease groups; CTR, control group; # , groups for CNV and protein levels analyses; FEV 1 , forced expiratory volume in 1 second; FEV 1 /FVC, Tiffeneau-Pinelli index; ratio of forced expiratory volume in 1 second and forced vital capacity; * , difference to the control group statistically significant at the level of p<0.00001.
It is widely accepted that metalloproteinases (MMPs) engaged in the COPD pathogenesis degrade matrix proteins (elastin, collagen) and the metalloproteinase 9 (MMP-9) plays a principal role in this process [6]. The protease is a type IV collagen-degrading enzyme, and its presence is required for adequate matrix processing and lung repair [7,8]. However, MMP-9 oversecretion may cause an unwanted degradation of lung tissue, and this may be one of the causes of COPD [6], probably due to the tobacco smoke stimuli [4]. The increased expression of MMP-9 may be associated with an increasing level of gene promoter activity. In particular, the -1562C/T polymorphism (rs3918242) in the MMP-9 gene promoter was found to be associated with MMP-9 expression, and the -1562T allele leads to higher transcription activity [9].
In this study, we evaluated the role of MMP-9 gene -1562C/T polymorphism, as well as MMP-9 protein and its complexes with TIMP levels, in COPD development in Polish patients.

COPD Patient and Controls
Group. Three hundred thirtyfive patients (248 males and 87 females) with COPD were enrolled in the study. All subjects underwent routine diagnosis including the spirometry result and FEV 1 /FVC ratio reduction below the lower limit of the norm. The spirometry test was performed twice, before the bronchodilator application (400 g of salbutamol) and after (15 -20 minutes). The outcome of the bronchial relaxation trial performed in that way should be negative. Patients with alpha-1-antitrypsin deficiency, coexistence of asthma, and COPD or with earlier history of bronchial asthma as well as patients who have never smoked were excluded from the study. All patients were recruited from the inpatient and outpatient populations of the University Hospital in Cracow, Specialized Hospital of Ministry of Internal Affairs and Administration (MSWiA) in Głuchołazy and Department of Pulmonology, Opole Voivodeship Hospital.
Three hundred and nine unrelated healthy volunteers (80 men and 229 women) not diagnosed for COPD nor any other lung disease and exhibiting normal lung function (mean FEV 1 % of 91.7%) served as a control group. To determine the effect of an exposure to tobacco smoke on analyzed factors, the controls were divided into subgroups of those with tobacco smoking history (110 current and former smokers) and those who had never smoked (199 never smokers).
For CNV and protein levels examination, 60 patients with COPD and 61 control individuals (23 smokers and 38 never smokers) were randomly selected.
All members of the patients and control group were of Polish Caucasians ethnicity, and detailed characteristics are shown in Table 1.
The project was approved by the Ethics Committee of Opole Voivodship. Signed informed consent was obtained from all persons tested.

DNA Isolation and SNP and CNV
Genotyping. gDNA was extracted from whole blood using the GeneMA-TRIX Quick Blood DNA Purification Kit (EURx, Poland) following the manufacturer's instructions. The -1562C/T polymorphism of the MMP-9 gene (rs3918242) was typed by the PCR-RFLP method as described previously [9]. Briefly, polymerase chain reactions were carried out in 20 l of solution containing a standard PCR buffer, 200 ng of tested DNA, 1 U of Taq Polymerase (Invitrogen, Brazil), 2 mM MgCl 2 , 200 M dNTPs (Invitrogen, Brazil), and 1 M of each primer (forward: 5 -GCCTGGCACATAGTAGGCCC-3 and reverse: 5 -CTTCCTAGCCAGCCGGCATC-3 ). Amplifications were performed under the following conditions: 2 min at 94 ∘ C; 30 cycles of 30 s at 94 ∘ C; 30 s at 60 ∘ C; 60 s at 72 ∘ C with the last elongation step of 7 min at 72 ∘ C. The PCR product was digested with 1 U of SphI restriction enzyme (Fermentas, USA) for 3 h at 37 ∘ C (435 bp amplicons containing a T allele were cut into two fragments of 244 bp and 191 bp). All digested products were electrophoresed on 3% agarose gel and visualized in a UV transilluminator using ethidium bromide staining.
The CNV polymorphism was typed using the quantitative polymerase chain reaction (qPCR) by TaqMan Copy Number Assays (Applied Biosystems of Life Technology, Foster City, CA) according to the manufacturer's protocol in total reaction volume of 20 L (contained 20 ng gDNA, 1x probes for both the target (hs 00238040cn) and reference gene (RNase P) and 1x TaqMan Genotyping Master Mix). The PCR program was processed as follows: 95 ∘ C for 10 min, 40 cycles of 95 ∘ C for 15 s, and 60 ∘ C for 1 min. Data were collected at the end of each cycle. All DNA samples were run in triplicate to ensure accurate results. Then, the CopyCaller Software (Applied Biosystems by Life Technology, CA) was used to analyze copy numbers of the tested gene in the samples.

Analysis of Protein Levels.
The serum level of MMP-9 was measured using the Quantikine enzyme-linked immunosorbent assay (ELISA), and serum concentrations of its complexes with TIMP1 and TIMP2 were determined by DuoSet ELISAs (all R&D, MN, USA). All analyses were performed according to the manufacturer's instruction using serum samples either nondiluted (for MMP-9) or diluted of 100-fold (for complexes). The absorbance was read with the Epoch microplate reader (BioTech, Winooski, VT, USA) at 450 nm as the primary wave length and 540 nm as the reference. Concentrations of proteins in samples were calculated with the equation of the standard curve using Gen5 software (BioTech, Winooski, VT, USA).

Statistical Analysis.
Deviation of the genotype counts from Hardy-Weinberg equilibrium was tested using the 2test and EpiInfo software. Differences in alleles and genotypes distribution between controls and patient groups were estimated using the two-tailed Fisher's exact test and 2test with Yate's correction, respectively. GraphPad InStat 3 software was used for distribution analyses. Moreover, the multiple inheritance models (codominant, dominant, recessive, overdominant, and log-additive) via logistic regression with binary response (a disease status) using SNPstats web tool (https://www.snpstats.net) were estimated. In protein levels, before relevant statistical comparison of all obtained data, the distribution of values was checked with the method of Shapiro-Wilk. One-way analysis of variance (ANOVA) was used to assess differences between obtained values of protein concentration. Subsequently, when significant interactions in ANOVA were identified, appropriate Tukey post hoc tests were applied. For the analysis of relationship between protein levels and lung function parameters, a multiple ridge regression model was used. For analysis of COPD development predictors, a logistic regression fitted with the disease status as a response variable was used and as covariates were selected: SNP rs3918242, CNV, gender, smoking status and levels of MMP-9, and its complexes with TIMP1 and TIMP2. These statistical analyses were performed using Statistica v.12 (StatSoft, Inc., OK, USA). In all analyses, p values ≤0.05 were considered significant.

Results
In total, 335 patients affected with COPD and 309 healthy individuals were typed for SNP rs3918242 in the promoter of the gene encoding MMP-9. The observed genotype frequencies were very close to the values expected for both cases and controls populations according to the Hardy-Weinberg equilibrium (p<0.05, data not shown). There were no significant differences in alleles and genotypes frequency distribution between the COPD patients and controls (Table 2). When controls were divided into two groups according to the smoking status, nonsignificantly lower -1562C allele frequency in smokers was observed (83.2% versus 87.1% in COPD group, p = 0.09; Table 2). Additionally, no differences between patients with COPD and controls in genotype frequencies distribution were observed in dominant (CC versus CT+TT), recessive (CC+CT versus TT), and overdominant (CC+TT versus CT) models of inheritance (Table 1S in Supplementary Material).
The numbers of MMP-9 gene copies were analyzed in the groups of 60 randomly selected patients with COPD and 61 healthy volunteers. We found that 85.0% of COPD patients and 82.0% of controls had 2 copies of the MMP-9 gene. Additionally, we also found individuals with 1 copy (3.3% and 4.9% in patients and controls, respectively), 3 copies (11.7% and 9.9% in patients and controls, respectively), and 4 copies (3.2% of controls). However, no significant difference in CNV frequency between COPD patients and the control group was found ( Table 2).
We also evaluated the levels of MMP-9 and its complexes with TIMP1 and TIMP2 in serum of COPD patients and controls (the same as selected for CNV) ( Table 3). We found that the mean serum MMP-9 levels in the COPD group were significantly higher in comparison with the control group (149.0 ng/ml versus 26.5 ng/ml; p < 0.0001), as well as those of the controls subgroups with smoking status (27.5 ng/ml in smokers and 25.9 ng/ml in never smokers, p = 0.37 for comparison between both control subgroups). In contrast, there were no significant differences in the mean serum levels of MMP-9/TIMP1 and MMP-9/TIMP2 between the COPD patients and controls, except a significant difference between COPD patients and total controls in levels of MMP-9/TIMP1 complex (3146.8 pg/ml versus 2970.1 pg/ml, p = 0.04). Additionally, serum of control smokers contained a significantly higher level of this complex in comparison to control nonsmokers (3135.8 pg versus 2869.8 pg, respectively; p = 0.03; Table 3).
In particular, in systematic comparison between SNP rs3918242, CNV genotypes, and serum MMP-9, MMP-9/TIMP1 and MMP-9/TIMP2 levels showed no statistically significant differences (Table 4). However, the COPD patients with 2 copies of the MMP-9 gene exhibited lower MMP-9 serum level in comparison to the combined group of patients with 1 or more than 2 copies of the gene (142.9 ng/ml versus 186.8 ng/ml, p = 0.09; Table 4).
Generally, COPD patients were characterized by significantly lower lung function parameters (FEV 1 % and FEV 1 /FVC) in comparison to controls (Table 1). This phenomenon continued to be observed after division of groups according to SNP rs3918242 and CNV genotypes. On the other hand, MMP-9 genotypes-related intragroup comparisons did not reveal any significant differences (Table 4).
In addition, interactions between lung function parameters and levels of MMP-9, MMP-9/TIMP1, and MMP-9/TIMP2 were analyzed using ridge regression modeling. In controls group, an increment in MMP-9 levels was significantly associated with lower FEV 1 % (when MMP-9 level increases by 1 unit, the FEV 1 % decreases by 0.86). The Table 2: Alleles and genotypes distributions of -1562C/T SNP of MMP-9 gene (rs3918242) and copy number variability of MMP-9 gene in COPD patient and healthy control groups.
In patients with COPD group, the model showed a significant negative relationship between MMP-9 levels and values of both lung function parameters (FEV 1 % decreases by 0.06 and FEV 1 /FVC decreases by 0.04 for increased by 1-unit protein level). Additionally, MMP-9/TIMP1 complex level was positively related to FEV 1 /FVC, which increases by 0.006 for every 1-unit increasing in the complex level (Table 5). Table 6 shows a logistic regression analysis of potential predictors of COPD development; the variables found to be significantly associated with the COPD in univariate logistic regression analysis were gender (OR = 8.16, 95% CI = 5.73-11.61, p < 0.0001), smoking (OR = 606.04, 95% CI = 83.96-4374.76, p < 0.0001), and MMP-9 protein level (OR = 1.10, 95% CI = 1.06-1.14, p < 0.0001). Taking these covariates with significant association in univariable analysis into multivariable analysis revealed that MMP-9 serum level was considered as a potential independent risk factor (OR = 1.12, 95% CI = 1.03-1.23, and p = 0.001).

Discussion
It is postulated that the action of MMPs can lead to tissue degradation and emphysema and provide physiological remodeling of lung tissue, but when misregulated, may be associated with COPD development and severity. The protease-antiprotease imbalance is at the focus point of COPD pathogenesis hypothesis [10]. A classic example of this phenomenon is deficiency of the serine protease inhibitor, 1antitrypsin, which is commonly known as a major genetic factor in emphysema development [11] but accounts for only 3% of COPD patients. Thus, it seems that the crucial role in the pathogenesis of COPD is played by other proteases and their specific inhibitors. The role played by MMP-9 and its inhibitors TIMP1 and TIMP2 seems to be particularly important [9,11]. MMP-9 is an enzyme secreted by several cell types, including neutrophils and airway epithelial cells, as well as macrophages, which can be involved in COPD pathogenesis.
In an attempt to resolve that issue, we performed analysis of genetic and physiological features of MMP-9 in COPD pathogenesis.
In genetic analysis, our results suggest that there is no association between alleles and genotypes of the SNP rs3918242 (-1562 C/T) and COPD susceptibility among Polish COPD patients. This finding is in accordance with other studies on the Caucasian population with the exception of non-Hispanic Whites and Hispanic veterans, in case of whom it was concluded that TT genotypes constitute a risk factor for COPD development [12]. In contrast, the -1562T allele seems to be associated with the disease in Asians, but with some ambiguity in Koreans the T allele seems to play a protective role (reviewed by Stankovic et al. [13]). Nevertheless, two meta-analyses showed that SNP rs3918242 is associated with the risk of COPD [14,15].
Despite this contradictory role of mentioned polymorphism [6,12,[14][15][16][17], this SNP is most widely analyzed SNP of MMP-9 gene and it is considered as the functional promoter polymorphism. When T allele is present, the higher promoter activity has been shown [9,13], and it also was shown that COPD patients with the CT genotype expressed a higher serum level of MMP-9 [18]. Our results did not support this finding: we confirmed the results obtained by other authors [19][20][21][22] that the MMP-9 level in serum of COPD patients is higher in serum than in healthy controls, but we found that the difference does not depend on the SNP rs3918242, as well as on the number of MMP-9 gene copies (Table 4). Thus, we hypothesize that the increased secretion of MMP-9 in COPD patients may be a result of the disease-specific lung microenvironment, and this phenomenon could be explained by an inducible character of this enzyme [4].
The MMP is not produced by resident pulmonary macrophages in healthy lung; in contrast, in the COPD-related lung microenvironment, immunocompetent cells are the main source of MMP-9 [23]. It is known that stimulation of neutrophils leads to release of MMP-9 [24] and the alveolar macrophages from COPD patients produce more MMP-9 than macrophages from healthy controls in response to either inflammatory stimuli or cigarette smoke [25]. Therefore, it is suggested that MMP-9 serum level is associated with the disease severity and it is likely that it negatively correlates with lung function parameters (FEV 1 %, FEV 1 /FVC) [20,26,27]. In a compliance with this indication, we found the inverse relationship between MMP-9 level and FEV 1 % in both groups (patients with COPD and controls) and additionally with FEV 1 /FVC in COPD patients. Because of this, it cannot be excluded that MMP-9 is a biomarker for the presence and clinical course of COPD. Moreover, our result suggests that the enzyme level possesses a capability to be an independent predictor of the disease development. In aspect of this finding, it should be kept in mind that COPD is particularly associated with smoking (the variable included to our regression analysis). It is known that polymorphonuclear leukocytes from healthy controls could be stimulated by cigarette smoke to secrete MMP-9 at a level similar to the one found in COPD patients [4], but we did not find the different concentrations of MMP-9 in smokers and nonsmokers subgroups of controls. Nevertheless, it cannot be excluded that elevated level of MMP-9 accompanied smoking. Therefore, our regression observations need to be replicated, in the same or other populations, in order to consider MMP-9 as the biomarker and/or the predictor of COPD to reflect the complex role of MMP-9 in COPD.
MMP-9 is inhibited by the tissue inhibitors of metalloproteinase (TIMP1) and so the imbalance between MMP-9 and TIMP1 ratio could be involved in COPD pathogenesis [28]. Herein, in complement of the MMP-9 and TIMP1 ratio analysis [28,29], we demonstrate that the concentration of the MMP-9/TIMP1 complex in the serum of COPD patients is higher than that in healthy controls, which corroborates the findings of other authors [30]. More complexes of MMP-9/TIMP1 could be delivered by increased secretion of MMP-9, but it was also found that COPD patients exhibited a significantly higher level of unbound TIMP1 ( [29,30] and our unpublished data). Additionally, Lo et al. identified that smokers who exhibit the airway hyperresponsiveness  N, number of individuals; COPD, chronic obstructive pulmonary disease groups; CTR, control group; MMP-9, metalloproteinase 9; MMP-9/TIMP1, complex of metalloproteinase 9 and tissue inhibitor of metalloproteinase 1; MMP-9/TIMP2, complex of metalloproteinase 9 and tissue inhibitor of metalloproteinase 2; CNV, copy number variation.
produce increased level of MMP-9 to TIMP1, and the authors concluded that this finding indicates a new predictor to identify smokers vulnerable to COPD [31]. Interestingly, our results suggest that the MMP-9/TIMP1 complex concentration seems to be dependent on cigarette smoking (controls smokers exhibit a significantly higher level than nonsmokers). Several limitations of the present study should be taken into account. A potential important weakness is the design of the study performed for protein levels analysis. We measured MMP-9 and its complexes with TIMP1 and TIMP2 only at the protein level in blood, but not in situ using either sputum or bronchoalveolar lavage, which should better reflect the phenomena occurring in the lungs. Nevertheless, no significant difference between serum and induced sputum concentration of MMP-9 was found in a previous study [32]. In addition, despite the fact that in our study groups gender mismatching occurred, no intergender difference in analyzed factors was observed ( Figures 1S-5S in Supplementary Material), which is in accordance with the study by de Torres et al. [33], who found similar level of MMP-9 in women and men. Nevertheless, it seems that COPD phenotype depends on sex and men have milder dyspnea and better quality of life than women with a similar degree of airflow obstruction [33]. On the other hand, as we showed male gender could be considered as potential risk factor for COPD development.

Conclusions
We found that patients affected with COPD were predisposed to produce more MMP-9 and MMP-9/TIMP1 complex than healthy individuals. This phenomenon is probably associated with the disease-related lung environment, but not with genetic features of MMP-9 gene. Moreover, to the best of our knowledge, this is the first study in which copy number variation of the MMP-9 gene in the aspect of COPD development was investigated.

Data Availability
Data are available in Institute of Physiotherapy, Faculty of Physical Education and Physiotherapy, Opole University of Technology, Proszkowska 76 PL-45-758 Opole, Poland.