Lidia Ibba-Manneschi

Overexpression of VEGF165b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

Rationale: Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF165 and antiangiogenic VEGF165b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA. Objective: We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc. Methods and Results: Here, we show that the endogenous antiangiogenic VEGF165b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF165b expression correlated with increased expression of profibrotic transforming growth factor-&bgr;1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF165b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF165b than healthy MVECs. Transforming growth factor-&bgr;1 upregulated the expression of VEGF165b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF165b and SSc-MVEC–conditioned medium inhibited VEGF165-mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF165b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc MVECs and could be ameliorated by treatment with recombinant VEGF165 and anti-VEGF165b blocking antibodies. Conclusions: In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.

Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis

•  Introduction ‐  Evidence for endothelial disease and loss of capillaries in SSc ‐  Angiogenesis and vasculogenesis •  Mechanisms and biomarkers of endothelial damage in SSc •  Mechanisms of defective angiogenesis in SSc •  Mechanisms of defective vasculogenesis in SSc •  Conclusions and perspectives on vascular treatment strategies

Telocytes express PDGFRα in the human gastrointestinal tract

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34‐positive/c‐kit‐negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c‐kit‐positive/CD34‐negative/platelet‐derived growth factor receptor α (PDGFRα)‐negative interstitial cells of Cajal (ICC) and the PDGFRα‐positive/c‐kit‐negative fibroblast‐like cells (FLC). As TC display the same features and locations of the PDGFRα‐positive cells, we investigated whether TC and PDGFRα‐positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c‐kit and CD34/c‐kit double immunolabelling was performed in full‐thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34‐positive. TC formed a three‐dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c‐kit‐positive and CD34/PDGFRα‐negative. In conclusion, in the human GI tract the TC are PDGFRα‐positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region‐specific roles.

The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis

Background Early endothelial cell (EC) activation/damage and profibrotic Th2-associated cytokines play a pivotal role in systemic sclerosis (SSc). Interleukin 33 (IL33) is a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor. IL33 is also a chromatin-associated transcriptional regulator in ECs. Objective To investigate the role of the IL33/ST2 axis in SSc. Methods Skin biopsies were obtained from 30 patients with SSc (15 early/15 late stage) and 10 healthy subjects. Lung, kidney, heart, oesophagus, stomach, placenta biopsies and bronchoalveolar lavage cells from patients with SSc and controls were also analysed. IL33/ST2 expression was investigated by immunohistology, confocal immunofluorescence microscopy, western blotting and RT-PCR. Results In skin biopsies from control subjects, constitutive nuclear IL33 protein expression was found in dermal ECs and keratinocytes, while ST2 was weakly expressed in ECs and fibroblasts. In skin biopsies from patients with early SSc, IL33 protein was downregulated or absent in ECs and epidermis while IL33 mRNA was normally expressed or even upregulated. Moreover, ECs, perivascular infiltrating mast cells, CD68-positive macrophages, CD3-positive T cells, CD20-positive B cells and activated fibroblasts/myofibroblasts exhibited strong ST2 expression. In skin biopsies from patients with late SSc, IL33 was constitutively found in most ECs while ST2 immunostaining was weaker. In early SSc, the loss of endothelial IL33 protein and the overexpression of ST2 involved all affected organs. Dermal and pulmonary fibroblasts showed IL33 expression in SSc. Conclusion IL33 and ST2 are abnormally expressed in SSc. In early SSc, upon EC activation/damage IL33 may be mobilised from ECs to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts.

Autologous mesenchymal stem cells foster revascularization of ischemic limbs in systemic sclerosis: a case report.

BACKGROUND Mesenchymal stem cells can differentiate into endothelial cells and participate in angiogenesis in adults. In experimental models of acute myocardial infarction, mesenchymal stem cells led to the recovery of cardiac function through the formation of a new vascular network. OBJECTIVE To describe treatment with intravenous infusions of expanded autologous mesenchymal stem cells in 1 patient with critical limb ischemia due to systemic sclerosis. DESIGN Case report. SETTING The rheumatology unit at the University of Florence, Florence, Italy. PATIENT A woman, aged 34 years, with systemic sclerosis who developed acute gangrene of the upper and lower limbs. INTERVENTION 3 intravenous pulses of expanded autologous mesenchymal stem cells. MEASUREMENTS Angiography, skin histopathology, and immunohistochemistry. RESULTS Areas of necrotic skin were reduced after the first mesenchymal stem-cell infusion. After the third infusion, angiography showed revascularization of the patients extremities. Skin section analysis revealed cell clusters with tubelike structures, and angiogenic factors were strongly expressed. LIMITATION Causality cannot be established by a single case. CONCLUSION In patients with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may foster the recovery of the vascular network, restore blood flow, and reduce skin necrosis. PRIMARY FUNDING SOURCE Fondazione Cassa di Risparmio di Pistoia e Pescia (partial funding).

Mechanisms of vascular damage in systemic sclerosis

Although being classified as autoimmune connective tissue disease, dominant components of the pathophysiology of systemic sclerosis (SSc) consists of mechanisms of vascular damage, which can occur early in the course of the disease. Amongst them are abnormal vasoreactivity, hypoxia, insufficient neoangiogenesis and direct damage of vascular and perivascular cells. They result in a decreased capillary blood flow, and subsequently in clinically overt symptoms such as Raynauds syndrome and fingertip ulcers. In addition, in active disease vascular pathology can affect various other organs, predominantly the lung, the kidney, the heart but also the gastrointestinal tract. Vascular pathology contributes also significantly to overall morbidity and mortality in SSc patients and reduces life expectancy by at least a decade. Fortunately, molecular biology has revealed a number of underlying pathways on the cellular and subcellular levels, including key factors of the aberrant function of (peri)vascular cells and autoimmune effector cells, the dysregulation of vasoconstrictive molecules and their receptors, the upregulation of intracellular signaling kinases and the altered balance of hypoxia-induced vascular growth factors. This increasing knowledge of vascular pathology in SSc has also resulted in novel therapeutic approaches ranging from endothelin antagonists to application of progenitor cells to counteract this aberrant vascular pathology and to support the repair of the dysfunctional vasculature.

A loss of telocytes accompanies fibrosis of multiple organs in systemic sclerosis

Systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of the skin and various internal organs. In SSc, telocytes, a peculiar type of stromal (interstitial) cells, display severe ultrastructural damages and are progressively lost from the clinically affected skin. The aim of the present work was to investigate the presence and distribution of telocytes in the internal organs of SSc patients. Archival paraffin‐embedded samples of gastric wall, myocardium and lung from SSc patients and controls were collected. Tissue sections were stained with Massons trichrome to detect fibrosis. Telocytes were studied on tissue sections subjected to CD34 immunostaining. CD34/CD31 double immunofluorescence was performed to unequivocally differentiate telocytes (CD34‐positive/CD31‐negative) from vascular endothelial cells (CD34‐positive/CD31‐positive). Few telocytes entrapped in the fibrotic extracellular matrix were found in the muscularis mucosae and submucosa of SSc gastric wall. In the muscle layers and myenteric plexus, the network of telocytes was discontinuous or even completely absent around smooth muscle cells and ganglia. Telocytes were almost completely absent in fibrotic areas of SSc myocardium. In SSc fibrotic lung, few or no telocytes were observed in the thickened alveolar septa, around blood vessels and in the interstitial space surrounding terminal and respiratory bronchioles. In SSc, the loss of telocytes is not restricted to the skin, but it is a widespread process affecting multiple organs targeted by the fibrotic process. As telocytes are believed to be key players in the regulation of tissue/organ homoeostasis, our data suggest that telocyte loss might have important pathophysiological implications in SSc.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro

Objective To characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis. The study also analysed whether the production/release of bioactive mediators by MSCs were affected by stimulation with cytokines found upregulated in SSc serum and tissues, and whether MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis. Methods MSCs obtained from five patients with early severe diffuse SSc (SSc-MSCs) and five healthy donors (H-MSCs) were stimulated with vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) or stromal cell-derived factor-1 (SDF-1). Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ1 and receptors TβRI and TβRII were evaluated by quantitative real-time PCR, western blotting and confocal microscopy. VEGF, SDF-1 and TGFβ1 secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. Results In SSc-MSCs the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared with H-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation and CXCR4 redistributed from the cytoplasm to the cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ1, TβRI and TβRII in SSc-MSCs. TβRII redistributed from the cytoplasm to focal adhesion contacts. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than H-MSCs. Experiments with blocking antibodies showed that MSC-derived cytokines were responsible for this potent proangiogenic effect. Conclusion SSc-MSCs constitutively overexpress and release bioactive mediators/proangiogenic factors and potentiate dermal MVEC angiogenesis.

Telocytes in Crohn's disease

Crohns disease (CD) is a relapsing chronic inflammatory disorder that may involve all the gastrointestinal tract with a prevalence of terminal ileum. Intestinal lesions have a characteristic discontinuous and segmental distribution and may affect all layers of the gut wall. Telocytes (TC), a peculiar type of stromal cells, have been recently identified in a variety of tissues and organs, including gastrointestinal tract of humans and mammals. Several roles have been proposed for TC, including mechanical support, spatial relationships with different cell types, intercellular signalling and modulation of intestinal motility. The aim of our study was to investigate the presence and distribution of TC in disease‐affected and ‐unaffected ileal specimens from CD patients compared with controls. TC were identified by CD34/PDGFRα immunohistochemistry. In affected CD specimens TC disappeared, particularly where fibrosis and architectural derangement of the intestinal wall were observed. In the thickened muscularis mucosae and submucosa, few TC entrapped in the fibrotic extracellular matrix were found. A discontinuous network of TC was present around smooth muscle bundles, ganglia and enteric strands in the altered muscularis propria. At the myenteric plexus, the loss of TC network was paralleled by the loss of interstitial cells of Cajal network. In the unaffected CD specimens, TC were preserved in their distribution. Our results suggest that in CD the loss of TC might have important pathophysiological implications contributing to the architectural derangement of the intestinal wall and gut dysmotility. Further functional studies are necessary to better clarify the role of TC loss in CD pathophysiology.

Increased serum levels and tissue expression of matrix metalloproteinase-12 in patients with systemic sclerosis: correlation with severity of skin and pulmonary fibrosis and vascular damage

Objective To determine serum concentrations and tissue expression of matrix metalloproteinase-12 (MMP-12) and their correlation with clinical features in patients with systemic sclerosis (SSc). Methods Serum MMP-12 levels from 72 patients with SSc and 42 healthy volunteers were examined by ELISA. Immunohistochemical expression of MMP-12 was analysed in skin biopsies from 20 patients with SSc and 13 healthy subjects and lung biopsies from three patients with SSc-related interstitial lung disease (ILD) and five controls. Results Circulating levels of MMP-12 were significantly increased in patients with SSc compared with healthy controls. Serum MMP-12 levels were significantly higher in both patients with limited cutaneous SSc and those with diffuse cutaneous SSc than in healthy controls, and correlated positively with the extent of skin involvement. MMP-12 levels were raised in SSc patients with ILD compared with patients without ILD, and correlated with severity of lung restriction. Increased serum levels of MMP-12 were also associated with the presence of digital ulcers and severity of nailfold capillary abnormalities. In contrast to almost undetectable MMP-12 expression in healthy skin, MMP-12 was strongly expressed in keratinocytes, dermal endothelial cells, fibroblasts/myofibroblasts and inflammatory cells in the skin of patients with SSc. Affected lung tissue from patients with SSc-related ILD showed strong MMP-12 expression in capillary vessels, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa, while faint expression was observed in normal lung tissue. Conclusions MMP-12 levels are increased in patients with SSc and are associated with severity of skin and pulmonary fibrosis and peripheral vascular damage.