Hypoxia induced interstitial transformation of microvascular endothelial cells by mediating HIF-1α/VEGF signaling in systemic scleroderma

Background: Endothelial mesenchymal transition (EndMT) is a key pathological event for vasculopathy, and is one of the early features and hallmarks of systemic scleroderma (SSc). It has been well-established that hypoxia contributes to EndMT. However, little is known about the effects of EndMT induced by hypoxia on the skin microvascular remodeling of SSc, as well as the underlying mechanism. Methods: Skin biopsy was performed for SSc patients and healthy controls, and skin tissues were collected for isobaric tags for the relative and absolute quantication (iTRAQ)-based proteomics and immunohistochemical test. Human microvascular endothelial cell line-1 (HMEC-1) cultured in hypoxic or normal conditions was treated by tamoxifen or bevacizumab. The expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF)-a, CD31, α-smooth muscle actin (α-SMA), VE-cadherin, and bronectin were detected at both the protein and mRNA level. Results: The iTRAQ-based proteomics indicated the signicantly upregulated HIF-1 signal in the skin tissues of SSc patients. The immunohistochemical results demonstrated the signicant downregulation of endothelial cell (EC) marker CD31 and the distinct positive staining of interstitial cell (IC) marker α-SMA at sites that lined the vessel lumens in skin tissues of SSc. Meanwhile, the positive staining of HIF-1α, which is a key transcription factor in response to chronic hypoxia, and VEGF-a were found to be diffusely distributed in SSc skin tissues. Consistent with these observations, HMEC-1 cells cultured under hypoxic conditions exhibited a signicant decrease in CD31 and VE-cadherin expression, alongside the marked increase in the expression of α-SMA and bronectin, as well as the distinct upregulation of HIF-1α and VEGF-a, when compared with those under Conclusion: These results suggest that the HIF-1α/VEGF signaling pathway has a critical role in mediating the effect of hypoxia-induced EndMT on the skin microvascular remodeling of SSc.


Background
Systemic sclerosis (SSc) is a rare connective tissue disease with high disability and mortality rate, featured by autoimmunity, a broad microvasculopathy, and the brosis of the skin and visceral organs [1].
Endothelial-to-mesenchymal cell transition (EndMT) has emerged as a key player in the pathogenesis of tissue brosis and bro-proliferative vasculopathy in various diseases, including cardiac and intestinal brosis, diabetic nephropathy, portal hypertension, and pulmonary arterial hypertension (PAH) [1,2].Furthermore, experimental evidence supports the role of EndMT in the lung tissues of patients with interstitial lung disease associated with SSc [3,4].In addition, cells in the intermediate stages of EndMT were identi ed in the dermal microvessels of both patients with SSc, and bleomycin-induced and urokinase-type plasminogen activator receptor de cient mouse models [5].That is, EndMT causally connects the two hallmarks of SSc: the persistent endothelial injury and the aberrant brogenesis.
In the pathogenesis of SSc, reduced capillary blood ow coupled with de cient angiogenesis leads to chronic hypoxia, enforcing a positive feed-forward loop for sustaining vascular remodeling, which further promotes the irreversible extensive tissue brosis [2].This indicates that chronic hypoxia is a prominent trait that contributes to vasculopathy and tissue brosis in SSc.In recent years, a large number of studies have con rmed that hypoxia could induce EndMT in vitro and in vivo [6,7,8].However, the process of EndMT induced by hypoxia in the pathogenesis of SSc remains unclear.
Hypoxia inducible factor-1α (HIF-1α) is a key transcription factor that responds to chronic hypoxia, and is presently considered as the "main regulatory factor" of the hypoxia environment [9].It has been reported that HIF-1α promotes brotic disease, and its implicated function includes the stimulation of excessive extracellular matrix (ECM), vascular remodeling and futile angiogenesis, with further exacerbation of the chronic hypoxia, the deterioration of the patho brogenesis, and EndMT [10,11].The excessive expression of HIF-1α has been detected in tissues of systemic sclerosis [12,13].In addition, vascular endothelial growth factor (VEGF) is the predominant proangiogenic factor regulated by HIF-1α in hypoxia-related diseases, and the remarkable upregulation of VEGF has been observed in SSc specimens [10,13].Recently, the inhibition of the HIF-1α/VEGF signaling pathway has been reported to reverse the anti-angiogenesis effects of chrysophanol [14].Nevertheless, it needs to be further veri ed whether hypoxia induces the EndMT in the pathogenesis of SSc dependent on HIF-1α/VEGF signaling.
The present study provides immunohistological evidence for verifying the EndMT in the dermal specimens of patients with SSc, and the excessive increase in expression of HIF-1α and VEGF-a.The investigators further presented that human microvascular endothelial cell line-1 (HMEC-1) cells cultured under hypoxic conditions exhibited the characteristics of EndMT, and the activation of both HIF-1α and VEGF-a.Finally, the inhibition of HIF-1α/VEGF signaling by tamoxifen and bevacizumab effectively inhibited EndMT.These observations provide strong support to the hypothesis that HIF-1α/VEGF signaling has a critical role in mediating the effect of hypoxia-induced EndMT on the skin microvascular remodeling of SSc.This novel mechanism may represent an important and novel therapeutic target for the complications of SSc-associated bro-proliferative vasculopathy and brosis.

Patients and tissue samples
Skin tissue samples obtained from eight patients with SSc and eight age and gender-matched healthy controls were investigated (Table 1).The tissue samples of patients and controls were obtained by skin biopsy, which was performed at the A liated Hospital of Southwest Medical University.The investigators only included patients who met the American College of Rheumatology (ACR)/European League Against Rheumatism(EULAR) 2013 classi cation criteria for SSc [15].The eligibility criteria excluded the following key conditions: (1) aged <18 or >75 years old; (2) body mass index (BMI) >25; (3) patients with severe primary diseases, such as liver, kidney, hematopoietic system damage, and other skin and connective diseases; (4) patients with recent infection; (5) tumor patients.The control samples were obtained from the normal skin surrounding the nevus, when it was removed.All specimens were taken from the ngers, the back of the hand, and the forearm.All surgical biopsies were performed after the patient's provision of informed consent, and according to the Institutional Review Board-approved protocols of the A liated Hospital of Southwest Medical University.

Histopathology and immunohistochemistry
The quali ed skin tissue was xed with 10% formalin, embedded in para n and sliced into 4-μm sections.Slides that contained the para n-embedded skin tissue sections were histopathologically examined by staining with hematoxylin and eosin, and by immunohistochemical staining with the speci c EC and mesenchymal (myo broblast) cell markers.The procedure for the immunohistochemical staining was, as follows: After dewaxing and hydration, the specimens were washed with phosphate buffer saline (PBS) and repaired with antigen in citric acid buffer.Then, catalase blockers were added, and the antibodies were added.Next, the slides were initially incubated overnight with one of the following antibodies: anti-CD31 (1:50 dilution), anti-α-SMA (1:200 dilution), anti-HIF-1α (1:100 dilution), or anti-VEGF-a (1:100 dilution).The IgG binding was revealed after the incubation with the secondary antibody for one hour.Then, the slides were colored with the diaminobenzidine (DAB) chromogenic agent.Finally, the positive signals of each index were semi-quantitatively counted using ImageJ, and the analysis was carried out.

Extraction of total proteins from skin tissues
The skin tissues were taken from four patients and matched controls.These were immediately soaked in liquid nitrogen before storing at −80°C.Each tissue was grounded in lysis buffer using Tissue Lyser II at a frequency of 25 Hz for a period of 30 minutes.The skin tissues were chopped up with scissors, and an additional 30 minutes of sonication performed using the intelligent ultrasonic processor DH99-IIIN (Lawson, China) to lyse the tissues until total grinding.Then, these tissues were lysed in lysis buffer (4% sodium dodecyl sulfate, 0.1 M of dithiothreitol in 0.1 M of Tris-HCl, pH 7.6) at a ratio of 100 mg of tissue per 1 mL of lysis buffer.Afterwards, the tissues were vortexed and incubated for ve minutes at 95°C before being lysed for 20 seconds with one second OFF one second ON at 20% amplitude using the ultrasonic processor probe.Subsequently, the lysate was centrifuged at 14,000×g for 20 minutes at 4°C to remove the cell debris.Then, the supernatant was recovered in a new tube, and the amount of proteins was measured using the bicinchoninic acid (BCA) protein assay kit, according to manufacturer's instruction.The protein assay through the BCA method was used for the quanti cation of total proteins in the sample.

Absolute quantitation (iTRAQ)-based proteomics
An amount of 1 mg of protein from each sample was deposited in a ltration centricon Microcon YM-30 (Millipore).The washing steps were performed with 8 M of urea, 0.1 M of Tris-HCl, and pH 8.5 buffer.Then, the cysteine residues were blocked with 12 mM of methyl methanethiosulfonate for 30 minutes at room temperature.Next, the proteins were digested by trypsin with enzyme to a substrate ratio of 1:50 (w/w) at 37°C for 15 hours.Furthermore, the digested peptides were labeled with the iTRAQ Reagent Kit (Applied Biosystems, Foster city, CA, USA).The iTRAQ-labeled samples were analyzed using the NanoAquity UPLC system connected to the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scienti c, USA).All protein analyses were performed by Majorbio Bio-pharm Technology Co., Ltd.(Shanghai, China).The raw data les were analyzed using the Proteome Discoverer (Thermo Scienti c, Version 2.1) against the Homosapiens database (https://www.uniprot.org/taxonomy/9606).The MS/MS search criteria were, as follows: mass tolerance of 10 ppm for MS and 0.02 Da for MS/MS tolorance, trypsin was the enzyme with two missed cleavage allowed, the carbamido methylation of cysteine and the iTRAQ of the N-terminus and lysine side chains of peptides were the xed modi cation, and methionine oxidation was the dynamic modi cation.The false discovery rate (FDR) of peptide identi cation was set as FDR ≤0.01.A minimum of one unique peptide identi cation was used to support the protein identi cation.The functional enrichment analysis was performed for the identi ed proteins based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/).

Cell culture
The HMEC-1 cell line was obtained from Zhongqiao Xinzhou (Shanghai, China).The cells were cultured in endothelial cell medium (ECM, ScienCell) containing 5% fetal bovine serum (FBS), 1% penicillin/streptomycin double antibody, and 1% ECGS endothelial growth factor.Then, the cells were maintained at 37°C in a humidi ed atmosphere with 5% CO 2 .The anaerobic gas production bag (Hpebio, Qingdao, China) was used to promote the hypoxia environment, and this was placed into the anaerobic culture box.Then, the HMEC-1 cells were treated with hypoxia by culturing in the anaerobic culture box for 72 hours.The phenol blue oxygen indicator was used to monitor the oxygen concentration and hypoxia status.
Tamoxifen was dissolved in dimethyl sulfoxide (DMSO) to construct a solution at 5-mmol/L concentration.Then, this was ltered through a needle sterile lter, and stored at -20°C in the dark.These HMEC-1 cells could also be co-cultured with 5 umol/L of tamoxifen, with DMSO as the negative control.Bevacizumab was dissolved in PBS to construct a solution of 3-mg/ml concentration.Then, this was ltered through a needle sterile lter, and stored at -20°C in the dark.The HMEC-1 cells could also be cocultured with 0.3 mg/ml of bevacizumab, with PBS as the negative control.

Statistical analysis
All experiments were repeated for at least three times with similar results.The values were expressed as the mean ± SD of at least three independent experiments in triplicate.The statistical differences were assessed by one-way analysis of variance (ANOVA) (Kruskal-Wallis) using the SPSS 17.0 software.A P-value of <0.05 was considered statistically signi cant.

Background of participants and iTRAQ-based proteomics
The clinical features of the SSc patients and healthy controls, whose skin tissues were studied, are shown in Table 1.The protein patterns from four patients and four controls were analyzed using the iTRAQ-based quantitative approach.A total of 2,159 proteins were identi ed.For the amino acid sequences of the identi ed proteins, the protein dataset was matched to the local database, and 43 proteins were found to be signi cantly different between SSc patients and controls, with P <0.05, FC >1.2, or <0.83, as shown in Additional File 1.The functional enrichment analysis was performed for the identi ed proteins based on the KEGG database.The top 15 up and down pathways are shown in Figure 1A.Interestingly, the HIF-1 signal was signi cantly upregulated in the skin tissues of SSc patients.The EndMT and activated HIF-1α/VEGF signal were detected in patients with systemic scleroderma Compared with the controls, all eight skin tissue specimens obtained from SSc patients displayed varying degrees of interstitial brosis, along with a pattern of in ltration of in ammatory mononuclear cells around the vessels, marked narrowing, and occlusion of the vessel lumen and vascular loss, as illustrated in Figures 1B and 1C.EC marker CD31 and IC marker α-SMA were used in the immunohistologic analyses to identify the EndMT present in the cutaneous tissue.As expected, CD31 positive cells signi cantly decreased in SSc tissues, and these were detected at sites that lined the vessel lumens, when compared with those in control tissues, as illustrated in Figures 1F and 1G.Meanwhile, the positive α-SMA staining of α-SMA was perivascularly shown in the specimens of patients, and on the epidermal cells, but was negative in the control specimen, as illustrated in Figures 1H and 1I.
The HIF-1α/VEGF signal markers were also analyzed by immunohistochemistry.All skin biopsies obtained from SSc patients were both HIF-1α and VEGF positive.However, HIF-1α was not present, and VEGF was lowly expressed in the healthy tissue samples (Figs.1J, 1K, 1L and 1M).The staining of these two markers were observed throughout the keratinocytes of the epidermis, and this was diffusely distributed in the dermis.

Hypoxia induced the interstitial transformation of microvascular endothelial cells in vitro
In order to determine whether hypoxia is able to induce the conversion of ECs into ICs, the EC cell line HMEC-1 was exposed to an anaerobic gas production bag for hypoxia treatment, and the endothelial and brotic characteristics were measured.ECs exposed to anaerobic gas exhibited a decrease in mRNA expression for endothelial proteins VE-cadherin and CD31 (Figs. 2A and 2B) at every tested hypoxia time point.Furthermore, consistent with the establishment of the brotic process, ECs challenged with anaerobic gas exhibited an increase in mRNA level for brotic markers α-SMA and bronectin (Figs.2C and 2D) at every tested hypoxia time point.At the protein level, the expression level of CD31 and VEcadherin was temporarily upregulated after 24 hours of hypoxia, and gradually declined with the extension of hypoxia, as illustrated in Figure 2E, 2F and 2G.The protein of α-SMA and bronectin exhibited a continuous increase with the prolongation of the hypoxia treatment, as illustrated in Figures 2E, 2H and 2I.The immuno uorescence of cells revealed a similar trend, as illustrated in Additional File 2.
The effect of hypoxia on the cellular morphology and polarity of ECs was also investigated.In the absence of hypoxia, HMEC-1 cells presented as circular adherent cells.When these cells were anoxic for 12 hours, there was a small amount of death, but the shape was still full, and the outline was clear.With the duration of hypoxia, the number of cell death increased, and the morphology changed to fusiform, gradually losing the adhesion connection and polarization.The data is shown in Additional File 3.

Hypoxia activated the HIF-1α and VEGF signaling in vitro
Considering the postulation of the HIF-1α/VEGF signaling pathway, which is involved in the hypoxiamediated EndMT, the investigators were prompted to determine whether hypoxia induced the activation of signal molecules and their ligands at both the mRNA and protein level.There was no change in HIF-1α mRNA level at the different time points after hypoxia (Fig. 3A).However, ECs in the presence of hypoxia exhibited the increase in VEGF-a mRNA level at each of the tested time point (Fig. 3B).Meanwhile, the expression of the two ligands of VEGF, Vascular Endothelial Growth Factor Receptor-1 (VEGF-R1) and VEGFR2, were detected at different time points of hypoxia by real-time quantitative polymerase chain reaction (qRT-PCR).Finally, the activation of VEGF-R1 and the inhibition of VEGF-R2 were found, as illustrated in Figures 3C and 3D.At the protein level, the detection of HIF-1α and VEGF-a were performed by western blot, which indicated the upregulation of both molecules (Figs.3E, 3F and 3G).
The downregulation of HIF-1α inhibits the hypoxia-induced EndMT and VEGF signaling Although tamoxifen has been reported to be the speci c inhibitor of HIF-1α [9], further investigations are needed to determine whether HIF-1α signaling and its probable downstream VEGF signaling induced by hypoxia could be effectively abolished.To test this, hypoxia-treated ECs were incubated with tamoxifen for 72 hours, and signal molecules HIF-1α and VEGF-a were detected at the protein level.The results revealed that hypoxia-treated ECs with tamoxifen exhibited the sustained decrease in expression of both HIF-1α and VEGF-a (Figs. 4A, 4B and 4C).
Taking into account that hypoxia induces EndMT, and the expression of HIF-1α and VEGF-a, the investigators determined whether the activation of the HIF-1α/VEGF signaling is necessary for the EndMTinduced by hypoxia.Finally, the investigators used tamoxifen, the speci c inhibitor of HIF-1α/VEGF signaling, and determined the expression of CD31, α-SMA and bronectin in hypoxia-treated ECs.The results revealed that the decrease in mRNA expression of EC marker CD31 in hypoxia-treated HMEC-1 cells could be inhibited by tamoxifen intervention (Fig. 5A).Furthermore, hypoxia-treated ECs with tamoxifen exhibited a signi cant decrease in mRNA expression of brotic protein α-SMA and bronectin, which could upregulate in EC induced by hypoxia, without the inhibition of HIF-1α/VEGF signaling (Figs. 5B and 5C).The protein levels of CD31 and α-SMA were also detected by western blot.It was found that the expression of EC marker CD31 could not decrease, and even IC marker α-SMA was inhibited in hypoxia-treated ECs while co-culturing with tamoxifen (Figs.5D, 5E and 5F).
Hypoxia-induced EndMT is dependent on the activation of HIF-1α/VEGF signaling Although VEGF-a has been veri ed as the predominant proangiogenic factor regulated by HIF-1α in hypoxia-related diseases, it remains unknown whether the hypoxia-induced EndMT mediated by the upregulation of HIF-1α signaling was dependent on the activation of VEGF-a.Bevacizumab, the inhibitor of VEGF-a, was used to treat ECs in hypoxia conditions for 72 hours, and HIF-1α, VEGF-a, CD31 and α-SMA were detected at the protein level [16].VEGF-a could be gradually inhibited by its inhibitor, which could not intervene with the activation of HIF-α induced by hypoxia (Figs.6A, 6B and 6C).The downregulation of CD31 and upregulation of α-SMA induced by hypoxia could be perfectly rescued by the inhibition of VEGF-a (Figs. 6D, 6E and 6F).

Discussion
Therapeutic improvements have been reported to alleviate the relative damage of SSc complications on patient prognosis.However, previous articles did highlight a high mortality rate in SSc patients and SSc vasculopathy, in terms of pulmonary PAH, heart involvement, and scleroderma renal crisis, which still represents the rst cause of disease-related mortality [17][18][19][20].Although the mechanism of SSc vasculopathy is not fully understood, increasing evidence indicates that endothelial injury and subsequent endothelial dysfunction is a primary event that triggers the subsequent formation of typical vascular lesions [21].In clinical practice, Raynaud's phenomenon (RP), the most common symptom and clinical sign of the disease, is just the result from endothelial injury, an important initiating event in SSc [22][23][24].In this study, seven of eight SSc patients clinically manifested as RP, but none of them had digital ulceration, even gangrene, which involved 70% of European SSc cases at the end of the 10-year observation period [25].The short duration of the disease (1.04±0.82)for patients included in this study could partly explain the absence of symptoms of digital ulcer.Meanwhile, the decreased staining of CD31 and increasing expression of α-SMA around dermis vessels in the specimen of these patients suggested the developing EndMT for them.It is known that EndMT, a key player in the remodelling of injured vessels, may be reversible, which further accounts for the clinical results, showing that none of the irreversible ischemic tissue injury was associated to ulceration in this study, and this also possibly offers novel cues for treatment [2].
In addition, it has been proposed that the SSc-related EndMT process have differential pathogenetic roles depending on the type of affected vessels [26,27].In arterioles and small arteries, EndMT may lead to an accumulation of pro brotic myo broblasts in the vessel intima and media, thereby contributing to vessel remodeling, and clinically manifesting as digital ulcers and gangrene of the extremities.When affecting capillary vessels, EndMT may lead to the increase in the number of perivascular myo broblast, thereby contributing to tissue brosis and a parallel loss of endothelial cells, characterized by microvessel rarefaction, which clinically promote the nailfold video-capillaroscopy abnormalities and dermal brosis [27].In this study, most of the patients suffered from clinical RP, high titer of antinuclear antibody (ANA), strongly positive expression of anti scl-70 antibody, and pathological skin brosis, along with perivascular in ammation, intimal hyperplasia and vascular loss, while very few cases had pathologically occlusive vasculopathy such as obliteration of the lumen and microthrombi.
None of these patients had digital ulcer.Meanwhile, the decrease in staining of CD31 and increased expression of α-SMA were shown to be mainly around the dermis vessels in the specimen of patients with SSc, and the positive HIF-α and VEGF signaling were diffusely distributed in the dermis.These ndings support the importance of EndMT in skin microangiopathy during the active progression of SSc, and to which the HIF-α/VEGF signaling may contributed much.
It is common knowledge that EndMT refers to the trans-differentiation process, during which ECs downregulate the expression of its speci c EC markers, such as CD31 and VE-cadherin, acquiring a mesenchymal phenotype, which is characterized by the expression of α-SMA, bronectin, type I collagen, and others [26].Meanwhile, ECs gain a spindle-shaped broblast-like morphology, disaggregate, and lose polarity during this transition [26], which has been well-observed in HMEC-1 cells intervened by hypoxia in this study.Together, the decreased expression of CD31 and the upregulation of α-SMA could also be detected by both RT-PCR at the mRNA level and western blot and immuno uorescence at the protein level for hypoxia-treated HMEC-1 cells, which was consistent with the ndings in scleroderma tissue.These accumulated results testi ed that HMEC-1 cells are the reliable cell model for hypoxia-induced EndMT, although highly proliferative SV40-transformed cells sometimes do not differentiate into stable phenotypes as primary endothelial cells at the same time.
Previous studies have con rmed that hypoxia could induce EndMT in various diseases, including scleroderma, which contributes to the vicious circle of ongoing pathology, and is considered as an important hallmark in SSc patients [6][7][8][28][29][30].The Galectin-3, Endothelin-1 (ET-1), HIF-1α/Twist1, BMP-7/Smads and TGFβ1/SMAD signaling pathways have been proven to be critical mediators in PAH by regulating EndMT, and have a critical role in mediating the effect of hypoxia-induced EndMT in pulmonary arterial and cardiovascular remodeling [6,8,29,30].In addition, ET-1 is another important factor for promoting the EndMT process on both endothelial cell lines and primary ECs isolated from SSc patients [31][32][33].However, the role of HIF-1α/VEGF signaling in inducing EndMT was rst discussed in the present study throughout literatures.HIF-1α is the master transcriptional regulator of the adaptive response to hypoxia, which is the tightly regulated form of HIF-1, and will quickly be hydroxylated and degraded in normoxic conditions by prolyl hydroxylases [34][35][36][37].However, in hypoxia conditions, the expression of HIF-1α would dramatically upregulate [38,39].HIF-1α has been postulated to be dysregulated in various pathologic conditions, which was also detected throughout the keratinocytes of the epidermis in all skin biopsies obtained from scleroderma patients [13,40,41].In the present study, the positive staining of HIF-1α was not only throughout the keratinocytes of the epidermis, but also diffusely distributed in the dermis, which further hints the important role of hypoxia in the pathogenesis of SSc.In addition, HIF-1α has no effect on the change of mRNA.Hypoxia inactivates HIF-1α hydroxylase, which inhibits ubiquitin degradation, and this mainly occurs at the protein level.
VEGF has been reported to be signi cantly upregulated in all stages of brosis and dendritic endothelial cells, and was considered to be one of the main transcriptional targets of HIF-1α in hypoxia-related diseases [13,42].Traditionally, VEGF is identi ed as a key mediator of angiogenesis, which induce the differentiation, proliferation, and migration of endothelial cells, consequently contributing to the formation of vessels through both angiogenesis and vascular remodeling [42].VEGF exerts its biological functions by binding to its receptors, that is, tyrosine kinase receptors VEGFR1 and VEGFR2.However, unexpectedly, skin tissues of SSc patients with characterized vessel obstruction and loss demonstrate the strong upregulation of VEGF and its two receptors, with a more intense staining for VEGFR-2 than for VEGFR-1 [13,38].Furthermore, the increase in VEGF and HIF-1α, accompanied by the characteristics of EndMT, were observed in the skin of SSc patients in both previous articles and in the present study.Meanwhile, HMEC-1 treated by hypoxic exhibited the downregulated level of CD31, VE-cadherin expression, and there was a marked increase in the expression of α-SMA and bronectin, along with the increase in expression of HIF-1α and VEGF-a.It is noteworthy that the hypoxia induced EndMT was effectively reversed by tamoxifen and bevacizumab, the inhibitor of HIF-1α/VEGF pathway.
The present study has limitations, such as the lack of validation in primary cells.Although highly proliferative SV40-transformed cells could not sometimes differentiate into stable phenotypes as primary endothelial cells did at the same time, the expression of all markers were detected by both RT-PCR at the mRNA level and western blot and immuno uorescence at the protein level, which were consistent with the ndings found in scleroderma tissues of patients.These accumulated results con rm the reliability of these results.Another limitation of the present study was that protein inhibitors are directly used, instead of employing gene-level interventions, such as gene silencing or overexpression.However, tamoxifen and bevacizumab have been widely used as effective inhibitors of HIF-α and VEGF, which requires less cost, when compared to genetic intervention.

Conclusion
Taken together, the results from the present study provides evidence that hypoxia is a crucial factor in inducing the conversion of ECs into ICs through an HIF-1α/VEGF dependent mechanism that consequently promotes skin microvascular remodeling and brosis in SSc.This information would be bene cial for designing novel and improved therapeutic strategies against the complications of SSc associated to bro-proliferative vasculopathy and brosis.
The Immunohistochemical staining of skin tissues for CD31 and α-SMA.In the tissue from patients similar to that in G and I, the presence of CD31-positive cells decreased and α-SMA-positive signaling around the vessels increased in the dermis, when compared to control tissue, similar to that in F and H. (J, K, L and M) The immunohistochemical staining of skin tissues for the HIF-1α/VEGF signal.The specimens from SSc from a patient were both HIF-1α and VEGF positive, similar to that in K and M, and HIF-1α was not present, and VEGF was lowly expressed in the normal tissue samples, similar to that in J and L.
iTRAQ-based proteomics, and the histopathologic and immunohistologic assessments of skin tissues obtained from patients with SSc.(A) The KEGG pathway enrichment analysis was carried out based on the protein-concentrated proteins using the independent development process of Majorbio Biopharm Technology Co., Ltd.Fisher's exact test was employed.With the adjusted P< 0.05, the KEGG pathway was considered to be signi cantly enriched.The KEGG enrichment diagram revealed the corresponding relationship between the target protein set, the annotation and enrichment of the KEGG pathway.The left side is the protein, and the right is the name of the KEGG pathway that enriched the target protein.Log2FC was used to show the upregulation and downregulation protein.The Z score value was employed to identify the upregulation or downregulation pathways.The Z score for the HIF-1 signaling pathway was 1.0.(B and C) Hematoxylin and eosin staining of skin tissues of SSc patients, indicating a large amount of brous tissue in the dermis and in ltration of in ammatory mononuclear cells around vessels, as well as narrowing, occlusion of the vessel lumen and vascular loss.(D and E) Original magni cation ×100.Negative controls for immunohistochemistry. (F, G, H and I)

Figure 3 The
Figure 3

Table 1 .
Clinical and demographic characteristics of SSc patients and controls