Fibrosis of oesophagus, lungs, heart, and kidney in the course of
systemic sclerosis (SSc) may lead to dysfunction of the above
organs or even patients death. Recent studies point out
the role of angiogenesis and fibrosis disturbances in the
pathogenesis of SSc. Heart fibrosis is one of the most important
prognostic factors in SSc patients. So, the aim of our study was
to examine cardiovascular dysfunction in SSc patients and its
correlation with serum levels of vascular endothelial growth
factor (VEGF), endostatin, and tissue inhibitor of
metalloproteinase 2 (TIMP2). The study group comprised 34
patients (19 with limited scleroderma (lSSc) and 15 with diffuse
scleroderma (dSSc)). The control group consisted of 20 healthy
persons, age and sex matched. Internal organ involvement was
assessed on the basis of specialist procedures. Serum VEGF,
endostatin, and TIMP2 levels were evaluated by ELISA. We found
cardiovascular changes in 15 patients with SSc (8 with lSSc and
7 with dSSc). The observed symptoms were of different characters
and also coexisted with each other. Higher endostatin serum
levels in all systemic sclerosis patients in comparison to the
control group were demonstrated (P < .05). Also higher serum
levels of endostatin and TIMP2 were observed in patients with
cardiovascular changes in comparison to the patients without
such changes (P < .05). The obtained results support the notion
that angiogenesis and fibrosis disturbances may play an important
role in SSc. Evaluation of endostatin and TIMP2 serum levels
seems to be one of the noninvasive, helpful examinations of heart
involvement in the course of systemic sclerosis.
Physical inactivity increases the risk of atherosclerosis. However, the molecular mechanisms of this relation are poorly understood. A recent report indicates that endostatin, an endogenous angiostatic factor, inhibits the progression of atherosclerosis, and suggests that reducing intimal and atherosclerotic plaque tissue neovascularization can inhibit the progression atherosclerosis in animal models. We hypothesize that exercise can elevate the circulatory endostatin level. Hence, exercise can protect against one of the mechanisms of atherosclerosis.
We examined treadmill exercise tests in healthy volunteers to determine the effect of exercise on plasma levels of endostatin and other angiogenic regulators. Oxygen consumption (VO2) was calculated. Plasma levels of endostatin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were determined using ELISA. The total peak VO2 (L) in 7 male subjects was 29.5 ± 17.8 over a 4–10 minute interval of exercise. Basal plasma levels of endostatin (immediately before exercise) were 20.3 ± 3.2 pg/ml, the plasma levels increased to 29.3 ± 4.2, 35.2 ± 1.8, and 27.1 ± 2.2 ng/ml, at 0.5, 2, and 6 h, respectively, after exercise. There was a strong linear correlation between increased plasma levels of endostatin (%) and the total peak VO2 (L) related to exercise (R2 = 0.9388; P < 0.01). Concurrently, VEGF levels decreased to 28.3 ± 6.4, 17.6 ± 2.4, and 26.5 ± 12.5 pg/ml, at 0.5, 2, and 6 h, respectively, after exercise. There were no significant changes in plasma bFGF levels in those subjects before and after exercise.
The results suggest that circulating endostatin can be significantly increased by exercise in proportion to the peak oxygen consumption under physiological conditions in healthy volunteers. These findings may provide new insights into the molecular links between physical inactivity and the risk of angiogenesis dependent diseases such as atherosclerosis.
Background: In systemic sclerosis (SSc) the lack of an angiogenic response to hypoxia may be due to inappropriate synthesis of angiogenic and angiostatic factors. Tissue kallikrein (t-kallikrein), regulating the kallikrein-kinin system and acting on the microcirculation, is a potent angiogenic agent, and kallistatin is its natural inhibitor.
Objective: To evaluate, in patients with SSc, t-kallikrein and kallistatin levels and their correlation with clinical features and measures of microvascular involvement.
Patients and methods: Serum levels of t-kallikrein and kallistatin (ELISA) and t-kallikrein skin expression (immunohistochemistry) were studied in patients with SSc, and evaluated for subset (dSSc or lSSc), clinical and immunological features, and microvascular involvement (ulcers, telangiectasias, nailfold videocapillaroscopy).
Results: Circulating levels of t-kallikrein were higher in SSc than in controls (p<0.001). T-kallikrein did not differ between lSSc and dSSc, although it was higher in lSSc than in controls (p<0.001).T-kallikrein levels were higher in patients with early and active capillaroscopic pattern than in those with late pattern (p = 0.019 and 0.023). Patients with giant capillaries and capillary microhaemorrhages had higher t-kallikrein concentrations than patients with architectural derangement (p = 0.04). No differences in kallistatin levels were detected between patients with SSc and controls, or between lSSc and dSSc. In early SSc skin, the presence of t-kallikrein was found in endothelial and in perivascular inflammatory cells, while no staining in skin of advanced SSc was detected.
Conclusion: T-kallikrein levels are increased in patients with SSc, particularly in lSSc, and are associated with early and active capillaroscopic patterns. T-kallikrein may play a part in SSc microvascular changes.
Microvascular injury, oxidative stress, and impaired angiogenesis are prominent features of systemic sclerosis (SSc). We compared serum markers of these phenomena at baseline and after treatment with nifedipine in SSc patients. Forty successive SSc patients were compared with 20 matched healthy subjects. All SSc patients stopped taking calcium-channel blockers 72 hours before measurements. Twenty SSc patients were also examined after 14 days of treatment with nifedipine (60 mg/day). Quantitative ELISA was used to measure the serum concentrations of vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), carbonyl residues, and advanced oxidation protein products (AOPP). The median concentrations of VEGF, sVEGFR-1, sVCAM-1, carbonyl residues, and AOPP were significantly higher in SSc patients than in healthy subjects at baseline. A correlation was found between VEGF concentration and carbonyl residue concentration (r = 0.43; P = 0.007). Nifedipine treatment led to a significant decrease in concentrations of sVCAM-1, carbonyl residues, and AOPP but did not affect concentrations of VEGF and sVEGFR-1. Nifedipine treatment ameliorated endothelium injury in patients with SSc, as shown by the concentrations of adhesion molecules and oxidative damage markers. The fact that VEGF and sVEGFR-1 concentrations were not changed whereas oxidative stress was ameliorated by nifedipine is consistent with the hypothesis that VEGF signalling is impaired in SSc. However, more experimental evidence is needed to determine whether the VEGF pathway is intrinsically defective in SSc.
nifedipine; oxidative stress; sVCAM-1; systemic sclerosis; VEGF
Pro-angiogenic factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] and anti-angiogenic factors (endostatin) play important roles in the progression of pancreatic cancer. The purpose of the present study was to investigate the knockdown effect by either VEGF or bFGF siRNA on the expression and secretion of endostatin in pancreatic carcinoma cells. Pancreatic carcinoma cell lines (sw1990, Panc-1 and PCT-3) were treated with VEGF and bFGF siRNA. The expression of VEGF, bFGF and endostatin in pancreatic carcinoma cell lines was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Secretion of endostatin was measured by enzyme-linked immunosorbent assay (ELISA). bFGF and VEGF siRNA significantly reduced the expression of bFGF and VEGF mRNA, respectively, but did not affect mRNA and protein expression of endostatin in pancreatic carcinoma cell lines. However, secretion of endostatin in PCT-3, Panc-1 and sw1990 cells was significantly inhibited by bFGF and VEGF siRNA. This study demonstrated that pro-angiogenic factors (VEGF and bFGF) differentially modulate expression and secretion of anti-angiogenic factors (endostatin). This result may have important implications in the anti-angiogenesis therapy in pancreatic cancer.
pancreatic carcinoma; bFGF; VEGF; siRNA; endostatin
Angiogenesis, the development of new capillaries, is involved in leukocyte ingress into the synovium during the development and progression of rheumatoid arthritis. Several soluble and cell surface-bound mediators including growth factors, cytokines, chemokines, proteolytic matrix-degrading enzymes, cell adhesion molecules and others may promote synovial neovascularization. On the other hand, endogenous angiostatic factors, such as angiostatin, endostatin, interleukin-4 (IL-4), IL-13, interferons and some angiostatic chemokines are also produced within the rheumatoid synovium, however, their effects are insufficient to control synovial angiogenesis and inflammation. Several specific and non-specific strategies have been developed to block the action of angiogenic mediators. The first line of angiostatic agents include vascular endothelial growth factor (VEGF), angiopoietin, αVβ3 integrin antagonist, as well as non-specific angiogenesis inhibitors including traditional disease-modifying agents (DMARDs), anti-tumor necrosis factor biologics, angiostatin, endostatin, fumagillin analogues or thalidomide. Potentially any angiostatic compound could be introduced to studies using animal models of arthritis or even to human rheumatoid arthritis trials.
Angiogenesis; Rheumatoid arthritis; Targeting; Angiogenic mediators; Angiostatic agents
Systemic sclerosis (SSc) is characterised by disturbed vessel morphology and an overproduction of vascular endothelial growth factor (VEGF). The VEGF gene located on chromosome 6p21.3 has several polymorphisms.
To test the hypothesis that disturbed angiogenesis may be related to the genetic background of the VEGF gene.
Materials and methods
EUSTAR centres included European Caucasian patients with SSc and matched controls with osteoarthritis. The VEGF gene was genotyped by polymerase chain reaction, followed by restriction enzyme analysis. The 634 C/T and 936 C/G mutations and an 18‐base pair insertion/deletion at −2549 of the VEGF promoter region were tested.
416 patients with SSc and 249 controls were included in the study population. Of the patients with SSc, 42% had a diffuse cutaneous subtype, 16% had increased pulmonary arterial pressure and 61% had decreased carbon monoxide diffusion capacity. The genotype frequencies in the patients with SSc and in controls were in Hardy–Weinberg equilibrium. The allele and genotype frequencies of the polymorphisms did not differ between patients with SSc and controls. No association was found between these polymorphisms and disease phenotypes.
This study shows that there is no association between the three selected functional VEGF polymorphisms and SSc.
We investigated the serum concentration of endostatin in 84 patients with multiple myeloma (MM) and in 13 healthy controls. The level of measured anti-angiogenic agent was correlated with the phase and stage of the disease, and most importantly with clinical and laboratory parameters depicting the disease activity (haemoglobin, creatinine, albumins, calcium, M-component, C-reactive protein, beta2-microglobulin, lactate dehydrogenase, stage of bone disease) as well as serum levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor and transforming growth factor-beta. The median serum level of endostatin in MM patients was 58 ng/ml and was statistically significantly higher than in the control group (median, 40 ng/ml; p=0.015). MM patients in phase I (at diagnosis) had higher levels of endostatin (median, 69 ng/ml) than those in phase II (plateau phase after treatment) (median, 49 pg/ml; p=0.044). We did not find any statistical correlation between the level of endostatin and stage of MM according to the Durie and Salmon system. The serum concentration of endostatin in MM patients with a normal level of albumins was significantly higher than in others with hypoalbuminaemia (median, 62 ng/ml versus 39 ng/ml; p=0.033). Also, patients with a normal value of lactate dehydrogenase had a higher concentration of endostatin than those with values >425 U/l (median, 70 ng/ml versus 39 ng/ml; p=0.019). We did not show any statistical correlation between the concentration of endostatin and level of haemoglobin, creatinine, calcium, C-reactive protein, beta2-microglobulin and stage of bone disease. We failed to find positive or negative correlations between the level of endostatin and vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor and transforming growth factor-beta. The concentration of endostatin did not influence the probability of survival in MM patients in our study. In conclusion, our data indicate that endostatin has a higher level in MM patients than in healthy controls. Highest values were stated in active phases of the disease (at presentation and in progression). Different clinical and laboratory parameters generally do not influence the concentration of endostatin (except albumins and lactate dehydrogenase).
To determine the molecular basis of corneal avascularity during wound healing and determine the role of angiogenic and antiangiogenic factors in corneal vasculogenesis.
The expression of proangiogenic factors (vascular endothelial growth factor [VEGF]; basic fibroblast growth factor [bFGF]; matrix metalloproteinase-2 [MMP-2]; and membrane-type 1-MMP [MT1-MMP]) and antiangiogenic factors (pigment epithelium–derived factor [PEDF]; angiostatin; restin; and endostatin) was analyzed in avascular corneas and in models of corneal neovascularization (bFGF pellet implantation, intrastromal injection of MT1-MMP cDNA, and surgically induced partial limbal deficiency).
Immunohistochemistry demonstrated the presence of antiangiogenic factors (PEDF, angiostatin, restin, and endostatin) and proangiogenic molecules (VEGF, bFGF, MMP-2, and MT1-MMP) in the cornea after wounding. Proangiogenic MMPs were upregulated in stromal fibroblasts in the vicinity of invading vessels following bFGF pellet implantation. Corneal neovascularization (NV) was also induced by intrastromal injection of MT1-MMP naked cDNA in conjunction with de-epithelialization. Partial limbal deficiency (HLD-) resulted in corneal NV in MMP-7 and MMP-3 knockout mice but not in wild type controls.
Corneal angiogenic privilege is an active process involving the production of antiangiogenic factors to counterbalance the proangiogenic factors (which are upregulated after wound healing even in the absence of new vessels). Our finding that the potent antiangiogenic factors, angiostatin and endostatin, are colocalized with several MMPs during wound healing suggests that MMPs may be involved in the elaboration of these antiangiogenic molecules by proteolytic processing of substrates within the cornea.
Systemic sclerosis (SSc) is a connective tissue disorder characterized by tissue hypoxia due to vascular changes and excessive fibrosis of the skin and internal organs. Damage to blood vessels and endothelium, as well as imbalance of vascular homeostasis, impairment of angiogenesis and vasculogenesis are observed in the course of the disease. The aim of the study was to investigate the pro-angiogenic factors angiogenin and SDF-1α in patients with SSc.
Material and methods
Serum samples were collected from 50 patients with dSSc (diffuse SSc) and lSSc (limited SSc) and from 38 patients used as a healthy control group. We explored: 1) how the serum concentrations of SDF-1α and angiogenin differ in the investigated groups; 2) the correlation among chemokines in SSc and the duration of the disease, Raynaud’s phenomenon, sclerosis of the skin and TSS (total skin score).
Patients with SSc showed statistically significantly higher serum angiogenin concentration and there was no correlation between duration of the disease and Raynaud’s phenomenon, skin sclerosis or TSS. There was also no difference or no correlation between serum level of SDF-1α and the investigated groups.
The increase in angiogenin concentration in the serum in patients with SSc may confirm endothelial damage caused by hypoxia and reduced vascular perfusion due to the course of SSc without contributing to compensatory revascularization.
systemic sclerosis; angiogenesis; vasculogenesis; angiogenin; SDF-1α
Disseminated intravascular coagulation (DIC) is characterized by platelet and neutrophil activation. Platelets are the major source of circulating vascular endothelial growth factor (VEGF). Endostatin, an anti-angiogenic factor, is a fragment of collagen that is released from the extracellular matrix via the active cleavage of neutrophil elastase, thereby increasing the circulating level of endostatin. Hypercoagulable conditions such as DIC may induce the release of VEGF and neutrophil elastase from the platelets and neutrophils.
We enrolled 240 patients who were clinically suspected of having DIC. Plasma levels of VEGF, endostatin, and neutrophil elastase were determined using commercial ELISA kits. Patients were diagnosed as having overt DIC if the cumulative International Society on Thrombosis and Haemostasis Subcommittee score was >5.
Overt DIC was diagnosed in 80 of the 240 patients. The circulating VEGF and neutrophil elastase levels gradually increased according to the severity of coagulopathy, as reflected by the DIC score. However, the circulating endostatin level did not change significantly according to the DIC score. We divided the patients into 2 groups: the non-cancer and cancer patient groups, to exclude the VEGF release from tumor tissues. Interestingly, in non-cancer patients, higher VEGF and neutrophil elastase levels were found to be significant diagnostic markers for overt DIC.
Our findings suggest that circulating VEGF and neutrophil elastase levels are laboratory markers reflecting coagulation activity. They are expected to be potential diagnostic markers of overt DIC, especially in non-cancer patients.
Vascular endothelial growth factor; Neutrophil elastase; Disseminated intravascular coagulation
Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.
Neurotrophins (NTs) are able to activate lymphocytes and fibroblasts; they can modulate angiogenesis and sympathic vascular function. Thus, they can be implicated in the three pathogenic processes of systemic sclerosis (SSc). The aims of this study are to determine blood levels of Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT-3) in SSc and to correlate them with clinical and biological data.
Serum samples were obtained from 55 SSc patients and 32 control subjects to measure NTs levels by ELISA and to determine their relationships with SSc profiles.
Serum NGF levels were higher in SSc patients (288.26±170.34 pg/mL) than in control subjects (170.34±50.8 pg/mL, p<0.001) and correlated with gammaglobulins levels and the presence of both anti-cardiolipin and anti-Scl-70 antibodies (p<0.05). In contrast, BDNF levels were lower in SSc patients than in controls (1121.9±158.1 vs 1372.9±190.9 pg/mL, p<0.0001), especially in pulmonary arterial hypertension and diffuse SSc as compared to limited forms (all p<0.05). NT-3 levels were similar in SSc and in the control group (2657.2±2296 vs 2959.3±2555 pg/mL, NS). BDNF levels correlated negatively with increased NGF levels in the SSc group (and not in controls).
Low BDNF serum levels were not previously documented in SSc, particularly in the diffuse SSc subset and in patients with pulmonary hypertension or anti-Scl-70 antibodies. The negative correlation between NGF and BDNF levels observed in SSc and not in healthy controls could be implicated in sympathic vascular dysfunction in SSc.
The serum concentration of two pro-angiogenic cytokines: basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1), and anti-angiogenic factor endostatin in the serum of 80 never treated B-cell chronic lymphocytic leukemia (CLL) patients and 27 healthy volunteers was measured using an enzyme linked immunosorbent assay. The serum levels of both bFGF and TGF-beta1 were found to be significantly higher in the CLL group (median 40.5 pg/ml and 38.6 ng/ml respectively) when compared to the control group (median 9.4 pg/ml and 18.9 ng/ml, respectively) (p<0.001). The levels of endostatin were not significantly different in CLL and control groups (median 12.3 ng/ml and 8.4 ng/ml, respectively) (p=0.09). In the group of CLL patients the level of bFGF was significantly higher in patients with progressive disease as compared with patients with stable disease (median 90.5 pg/ml and 40.5 pg/ml respectively) (p<0.001). Patients in Rai stage III and IV also had significantly higher levels of bFGF than patients in Rai stage 0-II (median 100.1 pg/ml and 29.3 pg/ml respectively) (p<0.001). The levels of both TGF-beta1 and endostatin were lower in patients in Rai stage III and IV (median 28.9 ng/ml and 9.1 ng/ml respectively) than in patients in Rai stage 0-II (42.8 ng/ml and 13.1 ng/ml respectively) (p<0.001 and p=0.002 respectively). The level of endostatin was also lower in the group of CLL patients with progressive disease (median 10.0 ng/ml) as compared to patients with stable disease (median 20.5 ng/ml) (p=0.008). In conclusion, the disturbance in the balance between pro- and anti-angiogenic factors may have an important influence on the course of CLL.
Syndecan-1 (CD138) is a transmembrane heparin sulfate proteoglycan
expressed on distinct stages of differentiation of B-lymphoid
cells. Its prognostic value in B-cell chronic lymphocytic
leukemia (B-CLL) has not been evaluated so far. The serum
concentration of sCD138 and some angiogenesis-involved cytokines:
vascular endothelial growth factor (VEGF), basis fibroblast growth
factor (bFGF), and endostatin were studied in 52 previously
untreated patients with B-CLL. We found that bFGF and sCD138
levels were significantly higher in B-CLL patients than in
controls. In patients with sCD138 level or endostatin level below
the median value the lymphocyte count was higher than in patients
with serum level of those cytokines above the median value. In
patients with progressive disease bFGF level was significantly
higher and sCD138 level significantly lower than in patients with
stable one. Moreover, high sCD138 level was associated with longer
lymphocyte doubling-free survival, and, on the limit of
statistical significance, a high endostatin level was associated
with shorter progression-free survival. We conclude that serum
sCD138 level is increased in early stage B-CLL patients and may
have a positive prognostic value as to the dynamics of the
Knowledge about biological factors involved in exercise-induced angiogenesis is to date still scanty. The present study aimed to investigate the angiogenic stimulus of resistance exercise with and without superimposed whole-body vibrations. Responses to the exercise regimen before and after a 6-week training intervention were investigated in twenty-six healthy male subjects. Serum was collected at the initial and final exercise sessions and circulating levels of matrix metalloproteinases (MMP) -2 and -9, Vascular Endothelial Growth Factor (VEGF) and endostatin were determined via ELISA. Furthermore, we studied the proliferative effect of serum-treated human umbilical vein endothelial cells in vitro via BrdU-incorporation assay. It was found that circulating MMP-2, MMP-9, VEGF and endostatin levels were significantly elevated (P<0.001) from resting levels after both exercise interventions, with higher post-exercise VEGF concentrations in the resistance exercise (RE) group compared to the resistive vibration exercise (RVE) group. Moreover, RE provoked increased endothelial cell proliferation in vitro and higher post-exercise circulating endostatin concentrations after 6 weeks of training. These effects were elusive in the RVE group. The present findings suggest that resistance exercise leads to a transient rise in circulating angiogenic factors and superimposing vibrations to this exercise type might not further trigger a potential signaling of angiogenic stimulation in skeletal muscle.
Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.
Systemic Sclerosis (SSc) is characterized by skin thickening, fibrosis and vascular obliteration. The onset and course are heterogeneous. Prominent features include autoimmunity, inflammation and vascular damage.
Aim of study:
To measure the level of serum Anti-Annexin V antibodies in SSc patients and to study its significance in relation to vascular damage in these patients.
Patients and methods:
Twenty patients with SSc (12 with diffuse SSc and 8 with the limited form) and 10 healthy age and sex matched volunteers as controls were all subjected to routine laboratory testing and immunological profiling including antinuclear, anti-Scl-70, anticentomere, anticardiolipin antibodies and anti-annexin V antibodies titres. Vascular damage was assessed by clinical examination and assessment of the disease activity score, nailfold capillaroscopy and colour flow Doppler of the renal arteries; Doppler echocardiography was used for assessing pulmonary hypertension.
Anti-annexin V antibodies were detected in 75% of patients. Comparisons between anti-annexin V in diffuse and limited subgroups showed no significance; however a statistically significant positive correlation was found between Anti-annexin V titre and the degree of vascular damage in SSc patients. Anti-annexin V increased significantly in patients with severe vascular damage in comparison with those less affected (15.3 ± 6.6 vs. 11.25 ± 3.6, P < 0.05). A significant positive correlation was found between Anti-annexin V titre and both the ACL titre (r = 0.79, P < 0.001) and the resistive index of the main renal artery (r = 0.42, P < 0.05).
Anti-annexin V antibodies were significantly present in sera of patients with SSc. Patients with more severe forms of vascular damage had higher titres of these antibodies. Anti-annexin V antibodies are a sensitive predictor of vascular damage in SSc and could serve as a useful parameter in discriminating patients with a higher risk of vascular affection from those without.
systemic sclerosis; anti-annexin V antibodies; vascular damage
Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.
Endostatin is a potent endogenous anti-angiogenic agent which inhibits tumour growth. A non-synonymous coding polymorphism in the Endostatin gene is thought to affect Endostatin activity. We aimed to determine the role of this Endostatin polymorphism in breast cancer pathogenesis and any influence on serum Endostatin levels in healthy volunteers. Endostatin protein expression on a breast cancer micro array was also studied to determine any relationship to genotype and to breast cancer prognosis.
The 4349G > A (coding non-synonymous) polymorphism in exon 42 of the Endostatin gene was genotyped in approximately 846 breast cancer cases and 707 appropriate controls. In a separate healthy cohort of 57 individuals, in addition to genotyping, serum Endostatin levels were measured using enzyme linked immunosorbant assay (ELISA). A semi-quantitative assessment of Endostatin protein expression on immunostained tissue micro arrays (TMA) constructed from breast cancer samples of patients with genotype data was performed.
The rare allele (A) was significantly associated with invasive breast cancers compared to non-invasive tumours (p = 0.03), but there was no association with tumour grade, nodal status, vascular invasion or overall survival. There was no association with breast cancer susceptibility. Serum Endostatin levels and Endostatin protein expression on the tissue micro array were not associated with genotype.
The Endostatin 4349A allele is associated with invasive breast cancer. The Endostatin 4349G > A polymorphism however does not appear to be associated with breast cancer susceptibility or severity in invasive disease. By studying circulating levels and tumour Endostatin protein expression, we have shown that any influence of this polymorphism is unlikely to be through an effect on the levels of protein produced.
To evaluate the effect of verteporfin photodynamic therapy (PDT) on endostatin with regard to expression of vascular endothelial growth factor (VEGF) in human choroidal neovascular membranes (CNVs) secondary to age‐related macular degeneration.
A retrospective review of an interventional case series of 68 patients who underwent removal of CNV. 29 patients were treated with PDT 3–655 days before surgery. 39 CNVs without previous treatment were used as controls. CNVs were stained for CD34, CD105, Ki‐67, cytokeratin 18, endostatin, E‐selectin and VEGF. “Predominance score of VEGF over endostatin” (mean) was defined as the difference between VEGF and endostatin staining scores.
In four CNVs treated by PDT 3 days previously, PS was significantly higher in the retinal pigment epithelium (mean = 2.5, p = 0.006) and stroma (mean = 2, p = 0.015) than in the control group (mean = 0). At longer post‐PDT intervals, PS was significantly decreased in the retinal pigment epithelium (mean = 0, p = 0.019) and stroma (mean = 0, p = 0.015). Proliferative activity was high (p = 0.023), but mostly related to inflammatory cells. PDT did not influence E‐selectin expression significantly.
VEGF predominance over endostatin early after PDT might contribute to enhanced angiogenic activity associated with recurrences. Strategies upregulating or replacing endostatin early after PDT might increase the effectiveness of PDT.
Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.
Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.
Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.
Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.
OBJECTIVES—Anti-endothelial cell antibodies (AECA) have been detected in 19 to 30% of patients with systemic sclerosis (SSc). The objective of this study was first to assess the role of a secondary Sjögren's syndrome (SS) in the occurrence of AECA in SSc. Secondly, we researched AECA in patients with primary SS, and investigated whether AECA were associated with vascular manifestations (Raynaud's phenomenon and vasculitis).
METHODS—IgG-AECA were tested by an ELISA method in serum samples from 50 patients with SSc (16 of them had also a secondary SS), 50 patients with primary SS, and 50 healthy controls.
RESULTS—AECA levels were significantly higher in patients with SSc or primary SS than in healthy controls (p < 0.01 and p < 0.01, respectively). In patients with SSc, AECA values were significantly higher in patients with secondary SS (p < 10−5). In patients with primary SS, AECA levels were significantly higher in patients with Raynaud's phenomenon (p < 0.01), but not in patients with vasculitis.
CONCLUSION—In patients with SSc, AECA are associated with a secondary SS. In patients with primary SS, AECA are associated with Raynaud's phenomenon, but not with vasculitis.
To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular superoxide dismutase (EC-SOD) in vitreous body and serum in patients with proliferative diabetic retinopathy (PDR), and investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. To investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model.
EC-SOD and VEGF concentrations in vitreous and serum samples from PDR and macular hole (MH) were measured by ELISA. The effects of EC-SOD on VEGF-induced proliferation, migration, and tube formation were evaluated using human umbilical vein endothelial cells (HUVECs). Moreover, the effects of EC-SOD on VEGF-induced proliferation and migration were evaluated in HUVECs and primary normal human retinal microvascular endothelial cells.
Intravitreal concentrations of EC-SOD were significantly higher (p<0.01) in PDR (58.0±23.8 ng/ml, mean±SD) than in MH (29.3±6.6 ng/ml). Intravitreal concentrations of VEGF were dramatically higher (p<0.01) in PDR (798.2±882.7 pg/ml) than in MH (17.7±15.5 pg/ml). The serum concentrations of EC-SOD and VEGF did not differ between the two patient groups. The vitreous concentrations of VEGF correlated with those of EC-SOD in all patients (rs=0.61, p<0.001). In HUVECs, EC-SOD at 100 ng/ml significantly suppressed VEGF-induced proliferation and tube formation, but not VEGF-induced migration.
EC-SOD was increased together with VEGF in the vitreous body from PDR patients, suggesting that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis.
Several systemic autoimmune diseases are associated with an increased prevalence of atherosclerosis which could not be explained by traditional risk factors alone. In systemic sclerosis (SSc), microvascular abnormalities are well recognized. Previous studies have suggested an increased prevalence of macrovascular disease as well. We compared patients with SSc to healthy controls for signs of early atherosclerosis by measuring intima-media thickness (IMT) of the common carotid artery in relation to traditional risk factors and markers of endothelial activation.
Forty-nine patients with SSc, of whom 92% had limited cutaneous SSc, and 32 healthy controls were studied. Common carotid IMT was measured by using B-mode ultrasound. Traditional risk factors for cardiovascular disease were assessed and serum markers for endothelial activation were measured.
In patients with SSc, the mean IMT (median 0.69 mm, interquartile range [IQR] 0.62 to 0.79 mm) was not significantly increased compared with healthy controls (0.68 mm, IQR 0.56 to 0.75 mm; P = 0.067). Also, after correction for the confounders age, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein cholesterol (P = 0.328) or using a different model taking into account the confounders age, HDL cholesterol, and history of macrovascular disease (P = 0.474), no difference in IMT was present between SSc patients and healthy controls. Plaques were found in three patients and not in healthy controls (P = 0.274). In patients, no correlations were found between maximum IMT, disease-related variables, and markers of endothelial activation. Endothelial activation markers were not increased in SSc patients compared with controls.
SSc is not associated with an increased prevalence of early signs of atherosclerosis.