Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis.

BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

Association of Bariatric Surgery With Vascular Outcomes.

Importance: Bariatric surgical weight loss is associated with reduced cardiovascular mortality; however, the mechanisms underlying this association are incompletely understood. Objectives: To identify variables associated with vascular remodeling after bariatric surgery and to examine how sex, race, and metabolic status are associated with microvascular and macrovascular outcomes. Design, Setting, and Participants: This population-based longitudinal cohort included 307 individuals who underwent bariatric surgery. Participants were enrolled in the bariatric weight loss program at Boston Medical Center, a large, multi-ethnic urban hospital, with presurgical and postsurgical assessments. Data were collected from December 11, 2001 to August 27, 2019. Data were analyzed in September 2019. Exposure: Bariatric surgery. Main Outcomes and Measures: Flow-mediated dilation (FMD) and reactive hyperemia (RH) (as measures of macrovascular and microvascular function, respectively) and clinical variables were measured preoperatively at baseline and at least once postoperatively within 12 months of the bariatric intervention. Results: A total of 307 participants with obesity (mean [SD] age, 42 [12] years; 246 [80%] women; 199 [65%] White; mean [SD] body mass index, 46 [8]) were enrolled in this study. Bariatric surgery was associated with significant weight loss and improved macrovascular and microvascular function across subgroups of sex, race, and traditional metabolic syndrome (mean [SD] pre- vs postsurgery weight: 126 [25] kg vs 104 [25] kg; P < .001; mean [SD] pre- vs postsurgery FMD: 9.1% [5.3] vs 10.2% [5.1]; P < .001; mean [SD] pre- vs postsurgery RH: 764% [400] vs 923% [412]; P < .001). Factors associated with change in vascular phenotype correlated most strongly with adiposity markers and several metabolic variables depending on vascular territory (eg, association of weight change with change in RH: estimate, -3.2; 95% CI, -4.7 to -1.8; association of hemoglobin A1c with change in FMD: estimate, -0.5; 95% CI, -0.95 to -0.05). While changes in macrovascular function among individuals with metabolically healthy obesity were not observed, the addition of biomarker assessment using high-sensitivity C-reactive protein plasma levels greater than 2 mg/dL identified participants with seemingly metabolically healthy obesity who had low-grade inflammation and achieved microvascular benefit from weight loss surgery. Conclusions and Relevance: The findings of this study suggest that bariatric intervention is associated with weight loss and favorable remodeling of the vasculature among a wide range of individuals with cardiovascular risk. Moreover, differences in arterial responses to weight loss surgery by metabolic status were identified, underscoring heterogeneity in physiological responses to adiposity change and potential activation of distinct pathological pathways in clinical subgroups. As such, individuals with metabolically healthy obesity represent a mixed population that may benefit from more refined phenotypic classification.

Perivascular Adipose Tissue Inflammation in Ischemic Heart Disease.

OBJECTIVE: There is growing recognition that adipose tissue-derived proatherogenic mediators contribute to obesity-related cardiovascular disease. We sought to characterize regional differences in perivascular adipose tissue (PVAT) phenotype in relation to atherosclerosis susceptibility. Approach and Results: We examined thoracic PVAT samples in 34 subjects (body mass index 32±6 kg/m2, age 59±11 years) undergoing valvular, aortic, or coronary artery bypass graft surgeries and performed transcriptomic characterization using whole-genome expression profiling and quantitative polymerase chain reaction analyses. We identified a highly inflamed region of PVAT surrounding the human aortic root in close proximity to coronary takeoff and adjoining epicardial fat. In subjects undergoing coronary artery bypass graft, we found 300 genes significantly upregulated (false discovery rate Q<0.1) in paired samples of PVAT surrounding the aortic root compared with nonatherosclerotic left internal mammary artery. Genes encoding proteins mechanistically implicated in atherogenesis were enriched in aortic PVAT consisting of signaling pathways linked to inflammation, WNT (wingless-related integration site) signaling, matrix remodeling, coagulation, and angiogenesis. Overexpression of several proatherogenic transcripts, including IL1ß, CCL2 (MCP-1), and IL6, were confirmed by quantitative polymerase chain reaction and significantly bolstered in coronary artery disease subjects. Angiographic coronary artery disease burden quantified by the Gensini score positively correlated with the expression of inflammatory genes in PVAT. Moreover, periaortic adipose inflammation was markedly higher in obese subjects with striking upregulation (≈8-fold) of IL1ß expression compared to nonobese individuals. CONCLUSIONS: Proatherogenic mediators that originate from dysfunctional PVAT may contribute to vascular disease mechanisms in human vessels. Moreover, PVAT may adopt detrimental properties under obese conditions that play a key role in the pathophysiology of ischemic heart disease. Graphic Abstract: A graphic abstract is available for this article.

FLI1 and ERG protein degradation is regulated via Cathepsin B lysosomal pathway in human dermal microvascular endothelial cells.

OBJECTIVES: Friend leukemia integration 1 and erythroblast transformation-specific, important regulators of endothelial cell homeostasis, are reduced in microvascular endothelial cells in scleroderma patients, and their deficiency has been implicated in disease pathogenesis. The goal of this study was to identify the mechanisms involved in the protein turnover of friend leukemia integration 1 and erythroblast transformation-specific in microvascular endothelial cells. METHODS: The effects of lysosome and proteosome inhibitors on friend leukemia integration 1 and erythroblast transformation-specific levels were assessed by Western blotting and capillary morphogenesis. The effect of scleroderma and control sera on the levels of friend leukemia integration 1 and erythroblast transformation-specific was examined. RESULTS: The reduction in the protein levels of friend leukemia integration 1 and erythroblast transformation-specific in response to interferon α or Poly:(IC) was reversed by blocking either lysosomal (leupeptin and Cathepsin B inhibitor) or proteosomal degradation (MG132). MG132, leupeptin or CTSB-(i) also counteracted the anti-angiogenic effects of Poly:(IC) or interferon α. Scleroderma sera reduced protein levels of friend leukemia integration 1 and erythroblast transformation-specific in comparison to control sera. Treatment with CTSB(i) increased the levels of friend leukemia integration 1 and erythroblast transformation-specific in a majority of serum-treated samples. CONCLUSIONS: Inhibition of cathepsin B was effective in reversing the reduction of friend leukemia integration 1 and erythroblast transformation-specific protein levels after treatment with interferon α or scleroderma sera, suggesting that targeting cathepsin B may have a beneficial effect in SSc vascular disease.

Evidence for a Derangement of the Microvascular System in Patients with a Very Early Diagnosis of Systemic Sclerosis.

OBJECTIVE: To investigate whether patients with a very early diagnosis of systemic sclerosis (VEDOSS) may already present circulating markers and in vitro signs of microvascular dysfunction. METHODS: Serum samples were obtained from 55 patients with systemic sclerosis (SSc), 25 patients with VEDOSS, and 55 matched healthy controls (HC). Serum levels of pan-vascular endothelial growth factor (VEGF) and soluble neuropilin-1 (sNRP-1) were measured by ELISA. Human dermal microvascular endothelial cells (H-MVEC) were cultured and stimulated with SSc, VEDOSS, and HC sera. Protein expression of NRP-1 was analyzed by Western blotting, cell proliferation by 5'-bromodeoxyuridine assay, migration capacity by wound-healing assay, and capillary-like tube formation by Matrigel assay. RESULTS: Serum levels of pan-VEGF were increased in patients with VEDOSS and SSc versus HC (p = 0.05 and p = 0.003, respectively). Serum levels of sNRP-1 were significantly reduced in patients with VEDOSS and SSc compared with controls (p = 0.012 and p = 0.027, respectively). NRP-1 expression was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.001 vs HC). Proliferation was reduced in H-MVEC stimulated either with VEDOSS or SSc sera in comparison with HC sera (p = 0.015 and p = 0.043, respectively). Wound healing was compromised in H-MVEC stimulated with VEDOSS and SSc sera versus HC sera (p < 0.01 for both). Capillarogenesis was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.01) and SSc sera (p < 0.001) compared with cells stimulated with HC sera. CONCLUSION: Similar to patients with SSc, patients with VEDOSS already present biological signs of endothelial dysfunction. Our data demonstrate that VEDOSS sera significantly modify endothelial cell behavior and impair the angiogenic potential of the microvascular system.

Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells.

BACKGROUND: Systemic sclerosis (SSc) is characterized by endothelial cell (EC) apoptosis, impaired angiogenesis and peripheral microvasculopathy. Soluble α-Klotho (sKl) is a pleiotropic molecule with multiple effects on ECs, including antioxidant and vasculoprotective activities. On the EC surface, sKl interacts with vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and transient receptor potential canonical-1 (TRPC-1) cation channel to control EC homeostasis. Here, we investigated whether sKl might act as a protective factor to improve angiogenesis in dermal microvascular endothelial cells (MVECs) from SSc patients (SSc-MVECs). METHODS: Wound healing assay was performed on healthy dermal MVECs (H-MVECs) challenged with sera from healthy controls or SSc patients with or without the addition of sKl. Capillary morphogenesis on Matrigel was assessed in H-MVECs and SSc-MVECs at basal conditions and treated with sKl, as well as in H-MVECs challenged with healthy or SSc sera in presence or absence of sKl. The expression of α-Klotho, VEGF165b, VEGFR-2, TRPC-1, Ki67 and active caspase-3 in H-MVECs and SSc-MVECs was investigated by western blotting. Immunostaining for α-Klotho was performed in H-MVECs and SSc-MVECs, and in healthy and SSc skin sections. RESULTS: Treatment with sKl effectively counteracted the inihibitory effects of SSc sera on wound healing ability and angiogenic performance of H-MVECs. The addition of sKl significantly improved angiogenesis and maintained over time capillary-like tube formation in vitro by SSc-MVECs. Stimulation of SSc-MVECs with sKl resulted in the upregulation of the proliferation marker Ki67 in parallel with the downregulation of proapoptotic active caspase-3. The expression of α-Klotho was significantly lower in SSc-MVECs than in H-MVECs. The expression of TRPC-1 was also significantly decreased, while that of VEGFR-2 and VEGF165b was significantly increased, in SSc-MVECs compared with H-MVECs. Challenge with sKl either significantly increased TRPC-1 or decreased VEGF165b in SSc-MVECs. Ex vivo analyses revealed that α-Klotho immunostaining was almost absent in the dermal microvascular network of SSc skin compared with control skin. CONCLUSIONS: Our findings provide the first evidence that α-Klotho is significantly decreased in the microvasculature in SSc skin and that sKl administration may effectively improve SSc-MVEC functions in vitro by acting as a powerful proangiogenic factor.

Decreased expression of neuropilin-1 as a novel key factor contributing to peripheral microvasculopathy and defective angiogenesis in systemic sclerosis.

OBJECTIVES: In systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is required for optimal VEGF-A/VEGFR-2 signalling. Here, we investigated the possible involvement of Sema3A/NRP1 axis in SSc. METHODS: Circulating Sema3A and soluble NRP1 (sNRP1) were measured in patients with SSc and controls. NRP1 and Sema3A expression in skin biopsies was evaluated by immunofluorescence and western blotting. NRP1 expression was assessed in SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs), and in SSc and control endothelial progenitor cell (EPC)-derived endothelial cells (ECs). The possible impact of transcription factor Friend leukaemia integration 1 (Fli1) deficiency on endothelial NRP1 expression was investigated by gene silencing. The binding of Fli1 to NRP1 gene promoter was evaluated using chromatin immunoprecipitation. Capillary morphogenesis was performed on Matrigel. RESULTS: Decreased sNRP1 levels in SSc were associated with active and late nailfold videocapillaroscopy patterns and digital ulcers. No difference in Sema3A was found between patients and controls. NRP1 was significantly decreased in SSc-MVECs both ex vivo and in vitro. NRP1 and Fli1 significantly decreased in H-MVECs challenged with SSc sera, while they were not different in SSc and control EPC-derived ECs. Fli1 occupied the NRP1 gene promoter and Fli1 gene silencing reduced NRP1 expression in H-MVECs. NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic capacity comparable to that of cells treated with SSc sera. CONCLUSION: In SSc, NRP1 deficiency may be an additional factor in the perturbed VEGF-A/VEGFR-2 system contributing to peripheral microvasculopathy and defective angiogenesis.

Plexin-D1/Semaphorin 3E pathway may contribute to dysregulation of vascular tone control and defective angiogenesis in systemic sclerosis.

INTRODUCTION: The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis. METHODS: Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynaud's phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides. RESULTS: Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs. CONCLUSIONS: Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.

Decrease of LL-37 in systemic sclerosis: a new marker for interstitial lung disease?

Interstitial lung disease (ILD) is the leading cause of systemic sclerosis (SSc) related morbidity and mortality. LL-37 peptide is the only cathelicidin of the human antimicrobial peptide family with antimicrobial effects and immunomodulatory activity. LL-37 has anti-fibrotic effects and anti-apoptotic effects on SSc dermal fibroblasts. The aim of the study was to investigate the circulating levels of LL-37 in SSc patients and its association with clinical, laboratory, and instrumental parameters. Fifty-eight SSc patients (30 with and 28 without pulmonary involvement) and 28 healthy controls were enrolled in the study. Pulmonary involvement was defined when ILD was found at HRCT (ground glass, reticular, and honeycombing pattern). Circulating LL-37 levels were measured with ELISA test. In SSc patients with ILD serum, LL-37 concentrations were remarkably lower (1.36 mg/ml) than those in SSc patients without ILD (4.62 ng/ml, p = 0.035) and controls (5.53 ng/ml, p = 0.009). In SSc patients without ILD, serum LL-37 levels were not different from controls (p = 0.812). No significant association or correlation was found between LL-37 levels and any other clinical, serological, or instrumental features. Serum LL-37 levels are significantly lower in patients with SSc ILD. Our results may suggest that lower LL-37 levels may be associated with the development of ILD. Whether circulating levels of LL-37 might be used as an indirect marker of ILD remains to be determined in larger SSc cohorts.

Vascular biomarkers and correlation with peripheral vasculopathy in systemic sclerosis.

Vascular disease is a hallmark of systemic sclerosis (SSc). It is present in every patient, being responsible both for the earliest clinical manifestations and the major life-threatening complications of the disease, and thus determining important morbidity and mortality. In SSc, progressive vascular injury leads to vascular tone dysfunction and reduced capillary blood flow, with consequent tissue ischemia and chronic hypoxia. These phenomena are often accompanied by abnormal levels of vascular factors. Microangiopathy in SSc may be easily assessed by nailfold videocapillaroscopy. The variety of derangements detected in the nailfold capillaries is accompanied by abnormal levels of different vascular mediators and appears to be the best evaluable predictor of the development of peripheral vascular complications, such as digital ulcers. The purpose of this review is to summarize in SSc the most relevant vascular biomarkers and the main associations between vascular biomarkers and capillaroscopic parameters and/or the presence of digital ulcers. Vascular biomarkers could become useful predictive factors of vascular damage in SSc, allowing an earlier management of vascular complications.

Phosphatonins: physiological role and pathological changes.

Maintenance of proper serum phosphate concentrations is required for healthy life, and critical for normal skeletal development and integrity. Several hormones and regulatory factors such as vitamin D, parathyroid hormone (PTH), and the phosphatonins (FGF-23, sFRP-4, MEPE) among others, may play a role only in the long-term regulation of phosphorus homeostasis.FGF23 is part of a previously unrecognized hormonal bone-parathyroid-kidney axis. Its synthesis and secretion by osteocytes are positively regulated by 1,25(OH)2D and serum phosphorus and negatively by the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), and the enzyme N-acetyl galactosamine trasferase 3 (PPGGalNacT3), encoded by GALnT3 gene, prevents its degradation.FGF23 requires Klotho protein as a coreceptor for high affinity binding to cognate FGF receptors (FGFRs). Mutations of any of FGF23, Klotho or GALnT3 genes can lead to a syndrome characterized by hyperphosphatemia, ectopic calcifications and recurrent long bone lesions with hyperostosis. Phosphatonin have been shown to be implicated in several common diseases involving kidney and mineral metabolism. FGF23 might also represent a promising putative marker for bone healing.

Polymorphisms of estrogen receptors: risk factors for invasive melanoma - a prospective study.

Cross-sectional studies have reported associations between a number of polymorphisms in the estrogen receptor alpha (ERα) gene and the body mass index, hypertension, coronary flow reserve, coronary atherosclerosis, and osteoporosis. There are currently no data examining the genetic polymorphisms of the ERα and estrogen receptor beta (ERß) genes in melanoma patients. The aims of this study were to investigate the associations of genetic polymorphisms of the ERα and ERß genes with melanoma risk. The study group consisted of consecutive patients who visited the Department of Dermatology of the University of Florence between March 2005 and July 2007 for surgical excision of melanoma. In our study, homozygosity for the wild-type alleles showed different results at the PvuII, XbaI, and AluI restriction sites. Only the AluI site showed a lower proportion of the A allele in the melanoma group compared to the control group; the P and X alleles were lower in the control group than in the melanoma group. The distribution of wild-type alleles is important because these alleles have a protective role in the expression of altered proteins, which involves the ERs in our case. Because of the phenotypic prevalence of the wild-type allele, the heterozygotes did not express the polymorphism. The homozygosity of the polymorphic-type alleles shows that a alleles are more frequent in the case group than in the control group, with proportions of 43.8 and 39.5%, respectively. These results suggest that a polymorphism at the AluIrestriction site correlates with a higher proportion of melanoma. Thus, the polymorphism of ERß could ascribe to a higher susceptibility to melanoma.