Uncontrolled Expression of Vascular Endothelial Growth Factor and Its Receptors Leads to Insufficient Skin Angiogenesis in Patients With Systemic Sclerosis
Systemic sclerosis (SSc) skin lesions are characterized by disturbed vessel morphology with enlarged capillaries and an overall reduction in capillary density, suggesting a deregulated, insufficient angiogenic response. It has been postulated that this phenomenon is due to reduced expression of the potent angiogenic factor vascular endothelial growth factor (VEGF). In contrast to this hypothesis, we demonstrate that the expression of both VEGF and its receptors VEGFR-1 and VEGFR-2 is dramatically upregulated in skin specimens of SSc patients throughout different disease stages. Interestingly, upregulation of VEGF was not mediated by hypoxia-inducible transcription factor-1 (HIF-1) as indicated by only a weak expression of the oxygen-sensitive α-subunit of HIF-1 in the skin of SSc patients. This was unexpected on measuring low Po2 values in the SSc skin by using a polarographic oxygen microelectrode system. Considering our observation that PDGF and IL-1β costimulated VEGF expression, we propose that chronic and uncontrolled VEGF upregulation that is mediated by an orchestrated expression of cytokines rather than VEGF downregulation is the cause of the disturbed vessel morphology in the skin of SSc patients. Consequently, for therapeutic approaches aiming to improve tissue perfusion in these patients, a controlled expression and timely termination of VEGF signaling appears to be crucial for success of proangiogenic therapies.
Systemic sclerosis (SSc) is a multiorgan disease characterized by widespread fibrosis, activation of immune cells, production of autoantibodies, and injury to vascular as well as microvascular structures.1 The earliest clinical symptoms of SSc relate to disturbances in the peripheral vascular system.2 Nailfold capillaroscopy shows a variety of morphological changes including enlarged capillaries, bushy capillary formations, microhemorrhages, and a variable loss of capillaries with or without avascular areas. These phenomena are often accompanied by increased markers of endothelial cell injury and endothelial cell activation.3
Despite the reduced capillary density, there is paradoxically no sufficient angiogenic response in the skin of patients with SSc.4 Tissue ischemia leads usually to the expression of angiogenic growth factors, which then initiate angiogenic sprouting by inducing vasodilatation, proliferation, and migration of endothelial cells and stabilization of the lumina to form new vessels.5
Among the angiogenic growth factors, vascular endothelial growth factor (VEGF) has been identified as a key mediator of angiogenesis. VEGF induces differentiation, proliferation, and migration of endothelial cells that contribute to the formation of vessels through both angiogenesis and vascular remodeling. VEGF exerts its biological functions by binding to the tyrosine kinase receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1). Deletion of VEGF as well as of its receptors lead to embryonic lethality due to vascular defects, further highlighting the complex and multiple functions of the VEGF/VEGF receptor axis for angiogenic processes (see review)5. The expression of VEGF is tightly controlled and stimulated under hypoxic conditions through binding of the transcription factor hypoxia-inducible transcription factor-1 (HIF-1), a heterodimeric transcription factor consisting of the oxygen-regulated α-subunit and the constitutively expressed β-subunit, to a 47-bp sequence located 985 to 939 bp upstream of the transcription initiation site.6
Considering the lack of a sufficient angiogenesis despite the reduction in the capillary density, we hypothesized that the expression of the potent proangiogenic growth factor VEGF might be downregulated in patients with SSc. In the present study, we therefore analyzed the expression and regulation of VEGF and its receptors in skin specimens from SSc patients. In addition, we addressed the impact of HIF-1α on VEGF expression in SSc patients.
Materials and Methods
For an expanded Materials and Methods, see the online data supplement available at http://circres.ahajournals.org. Characteristics of SSc patients are shown in online Table 1.
Patients and Skin Biopsies
Skin biopsies were obtained from 15 patients who met the American College of Rheumatology criteria for SSc.7 In all patients, biopsies were taken from clinically involved skin. In a subset of patients (n=7), biopsies were also taken from clinically noninvolved skin as assessed by skin scoring of an experienced examiner. Controls (n=6) consisted of biopsies from healthy volunteers. Experiments were approved by local ethics review committees, and written informed consent was obtained from all patients.
In Situ Hybridization
Plasmids containing the 517 bp human VEGF121 cDNA were kindly provided by H.A. Weich (Max-Planck-Institute, Bad Nauheim, Germany) and used as a template to design VEGF-A–specific riboprobes.8 Preparation of VEGF-A–specific riboprobes and nonradioactive in situ hybridization was performed as described elsewhere.9 Hybridized probes were visualized with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Measurement of Skin Oxygenation
Skin oxygenation was measured intradermally in 13 SSc patients and 5 healthy controls at the dorsal aspect of the forearm (midway between wrist and elbow) using a Po2 histograph (Eppendorf, Germany). Healthy controls were age- and sex-matched with the SSc patients.
Cell Culture and Hypoxic Induction
Fibroblast cultures were obtained from skin biopsies of affected skin of additional patients. Control fibroblast cultures (n=5) were obtained from healthy subjects. For exposure to hypoxia, fibroblasts were grown to 50% to 80% confluence and transferred into a hypoxic incubator (Forma Scientific) containing 1% O2 v/v (hypoxia, oxygen tension 7 mm Hg) or 20% O2 v/v (normoxia, oxygen tension 140 mm Hg).
Western Blot Analysis and Immunofluorescence
Cultured cells were removed from the hypoxic incubator and rinsed quickly with ice-cold PBS. After cell lysis, extraction of nuclear proteins, and transfer on nitrocellulose membranes, HIF-1α protein was detected using monoclonal mouse anti–HIF-1α mgc3 antibodies.10 Similarly, for immunofluorescence on cultured cells, monoclonal mouse anti–HIF-1α mgc3 antibodies were used. Bound antibodies were visualized using FITC conjugated goat anti-mouse antibodies.
In all stimulation experiments, total RNA was isolated using the TRIzol LS reagent (Gibco/Life Technologies) according to the manufacturer’s protocol. Reverse transcription and TaqMan real-time PCR specific for VEGF-A was performed as described recently.11 18S was used as an endogenous control. Results were quantified with the threshold cycle (CT) and the comparative CT method. All experiments as well as all real-time PCR measurements were performed in duplicates.
HIF-1α protein was detected as described recently using monoclonal mouse anti–HIF-1α antibodies (Novus).12 For Flk-1/VEGFR2, monoclonal mouse antibodies were used. Flt-1/VEGFR1 protein was detected using polyclonal rabbit antibodies (both from Santa Cruz Biotechnology). For double labeling experiments, monoclonal mouse anti-CD68-antibodies (clone PG-M1, Dako) were used. Serial sections of in situ hybridization and VEGFR immunohistochemistry were stained with polyclonal rabbit anti-von Willebrand-Factor antibodies (A 0082, Dako).
Stimulation With Platelet-Derived Growth Factor and Interleukin-1β
Cultured dermal fibroblasts were grown to confluence in 24-well plates and stimulated with recombinant platelet-derived growth factor-BB (PDGF-BB) at 10 and 40 ng/mL and with recombinant interleukin-1β (IL-1β) at 1 pg/mL, 10 pg/mL, and 100 pg/mL. Recombinant proteins were purchased from R&D Systems (Abingdon, UK). Costimulation experiments were performed with 40 ng/mL PDGF-BB and 100 pg/mL IL-1β.
Data are shown as median with range. The Mann-Whitney test was used for statistical analysis. A value of P<0.05 was considered statistical significant.
VEGF mRNA Is Upregulated in Skin Biopsies From Patients With SSc
Consistent with recent findings,13 a constitutive expression of VEGF could be detected in the epidermis of healthy controls (Figure 1A). Whereas in some healthy controls the expression of VEGF was limited to suprabasal layers (Figure 1A), others showed a more scattered pattern of expression throughout the epidermis. As assessed by scoring in randomly chosen high-power fields, the mean percentage of keratinocytes expressing VEGF was 14% (range 0% to 30%). None of the healthy controls showed an expression of VEGF in dermal cells.
In contrast and despite the lack of sufficient angiogenesis in the SSc skin, an upregulation of VEGF was found in affected skin biopsies from patients with SSc (Figure 1B). The mean percentage of keratinocytes expressing VEGF was increased to 50% (range 0% to 100%, P≤0.05 compared with healthy controls). In addition to the enhanced expression in the epidermis, an enhanced expression of VEGF could be detected in the dermis of 13/15 patients. VEGF was expressed by a variety of cell types including fibroblasts and endothelial cells and inflammatory cells (Figure 1C and 1D). For double labeling experiments, see online data supplement. Interestingly, biopsy specimens taken from clinically noninvolved skin from the same patients (n=7) showed the same expression pattern of VEGF in each patient as observed for involved skin biopsies.
Hypoxia Is Present in Skin Lesions of SSc Patients
Given the strong upregulation of VEGF in skin biopsies of SSc patients, we next searched for possible stimulators of VEGF in the SSc skin. Using a Po2 histograph to measure tissue oxygen levels, SSc patients with nonfibrotic skin at the site of measurement (forearm) did not show different levels of intracutaneous Po2 values when compared with healthy controls. SSc patients with involved (fibrotic) skin at the site of measurement, however, had strikingly lower levels of Po2 than healthy controls and SSc patients without fibrotic changes at the forearm (P<0.05; Table). The lower levels of oxygen in the involved skin were not caused by systemic parameters, because no differences between the groups were found for arterial oxygen saturation, hemoglobin content, blood pressure, and heart rate (data not shown).
Characterization of the HIF-1α Induction by Hypoxia in Dermal Fibroblasts
Because the transcription factor HIF-1 is the key molecule regulating molecular responses to hypoxia, we next aimed to characterize the expression of its oxygen-dependent α-subunit (HIF-1α) in cultured dermal fibroblasts under hypoxic conditions. Note that the oxygen concentration used in these experiments (1% O2) is equivalent to a Po2 value of 7 mm Hg, which is close to the 10% percentile measured in fibrotic skin areas of SSc patients. An expression of HIF-1α was found by Western blotting after 6 hours of hypoxic exposure, whereas no signal could be detected after shorter exposure periods as well as in normoxic fibroblasts (Figure 2A). The accumulation of HIF-1α protein was maintained during prolonged exposure to hypoxia for 12, 24, and 48 hours at a similar expression level (Figure 2B). Interestingly, when these experiments were performed under low-serum conditions, the accumulation of HIF-1α under hypoxic conditions was considerably lower than in conditions using 10% fetal calf serum. This effect was seen in both SSc and normal fibroblasts (Figure 2B).
The HIF-1α protein needs to be translocated from the cytosol into the nucleus to induce molecular responses to hypoxia via binding to its HIF-binding site present in the flanking regions of HIF-target genes.14 As analyzed by indirect immunofluorescence, HIF-1α was almost completely located in the nucleus in dermal fibroblasts as early as 6 hours after hypoxic exposure (Figure 2C). No expression of HIF-1α was found by immunofluorescence in cells cultured under normoxic conditions. Taken together, these data indicate that HIF-1α can readily be activated under the given parameters in cells derived from SSc patients.
Effects of Hypoxia on VEGF Levels and Expression of HIF-1α Protein in SSc Skin Biopsies
We analyzed next whether the observed upregulation of HIF-1α by hypoxia is accompanied by an induction of VEGF in cultured dermal fibroblasts from SSc patients. As expected, hypoxic exposure for 24, 48, and 96 hours led to increased levels of VEGF mRNA in all cultures compared with fibroblasts kept under normoxic conditions (Figure 3). This induction of VEGF mRNA was of statistical significance at all time points (P<0.05) in both SSc and normal fibroblasts.
The experiments mentioned earlier suggested strongly that the abundant expression of VEGF in the SSc skin is mediated by HIF-1α that per se might have been activated by low Po2 levels in fibrotic skin areas of SSc patients. To address this hypothesis, we analyzed the expression of HIF-1α protein by immunohistochemistry in skin specimens from SSc patients and healthy controls and compared them with the expression of VEGF in the same subjects.
In skin specimens from healthy controls, nuclear signals for HIF-1α were detected in the epidermis with a moderate to high expression in all subjects (Figure 4A). (For detailed semiquantitative analysis, see online Table 2.) VEGF showed a similar pattern of expression in serial sections from the same healthy controls (Figure 4C). This coexpression of HIF-1α and VEGF in healthy subjects indicates that the constitutive expression of VEGF in the epidermis might in fact be driven by HIF-1α. In parallel with the results for VEGF, no expression of HIF-1α could be detected in the dermis of healthy controls.
Surprisingly, the expression of HIF-1α protein in skin specimens from SSc patients was lower than in those from healthy subjects. In the epidermis, we found weak signals for HIF-1α in a limited number of keratinocytes, which contrasted with the abundant epidermal expression of VEGF in these biopsies (Figure 4B and 4D). In addition, the expression pattern of HIF-1α did not correlate with the expression pattern of VEGF. Consistent with this observation, no HIF-1α protein was detected in dermal cells of SSc patients, despite the strong upregulation of VEGF in the dermis. Taken together, these data suggest that HIF-1–independent mechanisms contribute to the upregulation of VEGF in the SSc skin.
Expression of VEGF in Dermal Fibroblasts
To compare the normoxic constitutive synthesis of VEGF in dermal fibroblasts from SSc patients and healthy controls, cells were grown to confluence and quantified for VEGF expression by real-time PCR. VEGF mRNA could be detected in all healthy and SSc fibroblast cultures with some variability between individual fibroblasts in both groups. In contrast to the observation in skin biopsies, no significant difference were detected between SSc and normal fibroblasts (median ΔCt normal: 8.2, range 6.2 to 8.6; median ΔCt SSc: 8.4, range 6.4 to 8.7). Similarly, under low growth factor conditions (1% FCS), no differences were found between fibroblasts derived from healthy controls and SSc patients (median ΔCt normal: 18.0, range 17.2 to 18.7; median ΔCt SSc: 17.8, range 17.3 to 19.1).
Cytokines Induce VEGF in Dermal Fibroblasts
Because HIF-1α does not appear to be a major mediator of VEGF upregulation in the SSc skin, we focused on PDGF as well as IL-1, both factors being implicated as key molecules in the pathogenesis of SSc in a number of previous studies.15–18 When fibroblasts were treated with recombinant PDGF, there was a dose-dependent increase of VEGF mRNA in both SSc and normal fibroblasts (10 ng/mL: normal, 1.6±0.4-fold increase; SSc, 1.6±0.3-fold increase; 40 ng/mL: normal, 3.0±0.8 fold increase; SSc, 1.8±0.5-fold increase; P<0.05 compared with nonstimulated controls).
Treatment with recombinant IL-1β resulted also in a dose-dependent increase of VEGF mRNA in all fibroblast cultures. Inductions were small but significant, reaching a 2.4±0.6-fold increase of VEGF mRNA in SSc fibroblasts and 1.9±0.2-fold increase in normal fibroblasts at concentrations of 100 pg/mL (P<0.05 compared with nonstimulated controls).
In vivo, SSc dermal fibroblasts are exposed to a variety of cytokines that are overexpressed in the SSc skin including PDGF and IL-1β.15–18 To mimic this situation, cultured cells were costimulated with recombinant PDGF and IL-1β and analyzed for VEGF mRNA levels. Whereas the stimulation with either PDGF or IL-1β resulted in a rather small increase of VEGF, the costimulation with 100 pg/mL IL-1β and 40 ng/mL PDGF showed additive effects with a strong increase of VEGF (Figure 5). The induction of VEGF raised to 4.7±0.5-fold increase in SSc fibroblasts compared with a 2.0±0.4 fold increase with IL-1β alone and a 2.3±0.2 fold increase with PDGF alone. These data indicate that the strong upregulation of VEGF in the SSc skin might be a net effect of different cytokines rather than an effect of a single factor.
Upregulation of VEGF Receptors in Systemic Sclerosis
Possible explanations for the lack of a sufficient angiogenesis in the SSc skin despite the upregulation of VEGF include a reduced expression of VEGF receptors (VEGFRs) on endothelial cells. To address this possibility, immunohistochemistry with antibodies against Flt-1/VEGFR1 and Flk-1/VEGFR2 was performed on skin sections of SSc patients and healthy controls. Expression of Flt-1/VEGFR1 was found in all biopsies from SSc patients (Figure 6A). VEGFR1 has been described as an endothelial specific molecule.19 Consistent with these reports, the expression of VEGFR1 was limited to endothelial cells of smaller vessels in the SSc skin (see also online Figure 3). In contrast to the significant expression in the SSc skin, expression of VEGFR1 could not be detected in 5/6 biopsies from healthy controls (Figure 6B), whereas one healthy control showed a weak expression on endothelial cells.
Similar to VEGFR1, the expression of Flk-1/VEGFR2 was found to be upregulated in SSc skin biopsies (Figure 6C). In agreement with the in situ hybridization for VEGF, VEGFR2 was found expressed in 13/14 patients. Again, the expression of VEGFR2 was preferentially detected on endothelial cells (see also online Figure 4). In general, the number of endothelial cells expressing VEGFR2 and the intensity of staining was higher than for VEGFR1. No other dermal structures than endothelial cells expressed VEGFR2, whereas some positive signals were observed in the epidermis from both SSc patients and healthy controls (Figure 6C and 6D). Interestingly, whereas in contrast to normal skin, both VEGF receptors were expressed also in biopsies from noninvolved (nonfibrotic) skin of SSc patients, the percentage of endothelial cells expressing both receptors was lower than in biopsies of involved (fibrotic) skin of the same patients (see online Tables 3 and 4 for detailed semiquantitative analysis of VEGFR1 and VEGFR2 immunohistochemistry).
In the present study, we addressed the hypothesis that the lack of sufficient angiogenesis in SSc might be explained by a downregulation of the potent angiogenic factor VEGF, which is a key molecule in several steps of angiogenesis.20 However, unexpectedly, skin specimens of patients with SSc showed a strong upregulation of VEGF by in situ hybridization compared with healthy controls. Apart from VEGF, also its receptors VEGF-R1 and VEGF-R were found to be upregulated on endothelial cells of SSc patients in vivo. In addition, we could show recently that VEGF protein was significantly increased in blood samples from patients with SSc,21 reaching levels observed in patients with numerous malignant diseases.22,23 Taken together, these data strongly imply an activation of the VEGF/VEGF-receptor axis in patients with SSc.
Angiogenesis is regulated by a tightly controlled balance of angiogenic and angiostatic factors.5 Thus, an attractive hypothesis for the insufficient angiogenesis in SSc despite upregulated increased levels of VEGF could be an upregulation of angiostatic factors that is even higher than those of VEGF and thereby outweighs its proangiogenic effects. However, data on angiostatic factors in SSc are inconsistent. For instance, the collagen type XVIII breakdown product endostatin has been described as significantly increased or not different from healthy controls.21,24 Recently, Macko et al25 found elevated levels of platelet factor-4 and thrombospondin-1 in plasma samples from patients with SSc.
Of particular interest regarding the role of VEGF in the pathogenesis of SSc is the increasing experimental evidence about the diverse biological actions of VEGF depending on the period and level of expression.26 Using pTET-VEGF165/MHCα-tTa transgenic mice, in which the expression of VEGF can be conditionally switched off in an organ-dependent manner by feeding tetracycline, Dor et al27 showed that overexpression of VEGF induces the formation of new functional vessels in adult organs. These vessels were mature as reflected by the presence of smooth muscle cells and resulted in an improved organ perfusion. However, most interestingly, prolonged exposure to VEGF without subsequently switching off its expression resulted in the formation of irregularly shaped sac-like vessels with reduced blood flow, very much reminiscent of the disturbed vessel morphology with megacapillaries seen in SSc patients. In fact, the timely downregulation of VEGF rather then the induction of angiostatic factors appears to be the major mechanism preventing the formation of chaotic vessel morphology. This is further evidenced by the association of chronic overexpression of VEGF with glomeruloid and hemangioma-like vessels in other experimental settings.28,29 Thus, although short-time upregulation of VEGF is a strong inducer of angiogenesis, chronic and uncontrolled overexpression of VEGF leads to chaotic vessel morphology with reduced blood flow in the newly formed vessels.
In the present study, overexpression of VEGF in the skin specimens was detected independent of the disease duration in both patients with early as well as late disease. In addition, recent detailed analysis of blood levels in SSc revealed that VEGF was significantly upregulated in patients with different disease durations including patients with very early stages that not yet fulfilled ACR criteria, but did so during a 1-year followup.21 Furthermore, cytokines known to induce the synthesis of VEGF are overexpressed in SSc throughout the disease course. In addition to PDGF and IL-1, this also includes TGFβ, which is considered a key factor in the development of fibrosis in SSc.30,31 Taken together, these data indicate that a chronic and uncontrolled overexpression of VEGF does occur in SSc and might significantly contribute to the chaotic capillary morphology seen in these patients. The uncontrolled overexpression of VEGF does already exist in earliest disease stages in parallel with the morphological capillary changes observed by capillary microscopy. The chronic expression appears to be driven by key cytokines in the pathogenesis of SSc including PDGF and IL-1 throughout different disease stages.
We also addressed other possible stimulators of VEGF in the SSc skin. Surprisingly, despite the clear presence of hypoxia in the involved skin of patients with SSc and despite the nuclear accumulation of HIF-1α in cells derived from SSc patients under hypoxic conditions in vitro, the expression of HIF-1α protein was lower in skin specimens from SSc patients than in healthy controls. Although in vitro the stabilization of HIF-1α protein is instantaneous32 and sustained for up to 48 hours in dermal fibroblasts, the response in vivo might be substantially different. Consistent with our findings, it has been reported that exposure of mice to 6% hypoxia leads to maximum levels of HIF-1α protein in the brain after 4 to 5 hours, but declines afterward, reaching basal normoxic levels after 9 to 12 hours.33 Similar results were obtained for the kidney and the liver. Considering the correlation of low oxygen levels with fibrotic structural changes in the skin of patients with SSc in the present study, hypoxia in SSc patients appears to be chronic rather than caused by acute situations such as vasoconstriction in the setting of Raynaud’s phenomenon. Whether the response of HIF-1α to chronic hypoxia is similar in the skin as reported for other organs has not yet been addressed. It has to be stressed, however, that our findings do not rule out that pathways induced by hypoxia independent from HIF-1α (eg, via induction of other members of the HIF family) significantly contribute to the pathogenesis of SSc.34 This is further supported by recent data showing that hypoxia directly contributes to the accumulation of extracellular matrix both by HIF-1α–dependent as well as HIF-1α–independent mechanisms.35
In summary, we have shown that, despite the lack of a sufficient angiogenesis in SSc, the proangiogenic factor VEGF together with its receptors VEGF-R1 and VEGF-R are upregulated in skin specimens from SSc patients. Although involved skin is characterized by strongly reduced levels of oxygen, hypoxia-induced HIF-1α does not appear to be the major mediator of VEGF induction in SSc. Instead, the chronic and uncontrolled overexpression of VEGF is based on a net effect of cytokines such as IL-1 and PDGF. SSc might therefore serve as a disease model for the effects of a chronic and unterminated upregulation of VEGF with the formation of irregularly shaped sac-like megacapillaries. Consequently, for therapeutic approaches aiming to improve tissue perfusion in these patients, a controlled expression and timely termination of VEGF signaling appears to be crucial for success of proangiogenic therapies.
The study was supported by the Deutsche Akademie der Naturforscher Leopoldina (grant BMBF-LPD 9901/8-7), the Braun Foundation, and grant 560031 of the University of Zurich. The authors thank Ferenc Pataky and Maria Comazzi for excellent technical assistance.
Original received December 1, 2003; revision received May 21, 2004; accepted May 24, 2004.
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