Irene Rosa

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.

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.

Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin‐embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Massons trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.

Inactivation of urokinase-type plasminogen activator receptor (uPAR) gene induces dermal and pulmonary fibrosis and peripheral microvasculopathy in mice: a new model of experimental scleroderma?

Objective Urokinase-type plasminogen activator receptor (uPAR) is a key component of the fibrinolytic system involved in extracellular matrix remodelling and angiogenesis. The cleavage/inactivation of uPAR is a crucial step in fibroblast-to-myofibroblast transition and has been implicated in systemic sclerosis (SSc) microvasculopathy. In the present study, we investigated whether uPAR gene inactivation in mice could result in tissue fibrosis and peripheral microvasculopathy resembling human SSc. Methods The expression of the native full-length form of uPAR in human skin biopsies was determined by immunohistochemistry. Skin and lung sections from uPAR-deficient (uPAR−/−) and wild-type (uPAR+/+) mice at 12 and 24 weeks of age were stained with haematoxylin-eosin, Massons trichrome and Picrosirius red. Dermal thickness and hydroxyproline content in skin and lungs were quantified. Dermal myofibroblast and microvessel counts were determined by immunohistochemistry for α-smooth muscle actin and CD31, respectively. Endothelial cell apoptosis was assessed by TUNEL/CD31 immunofluorescence assay. Results Full-length uPAR expression was significantly downregulated in SSc dermis, especially in fibroblasts and endothelial cells. Dermal thickness, collagen content and myofibroblast counts were significantly greater in uPAR−/− than in uPAR+/+ mice. In uPAR−/− mice, dermal fibrosis was paralleled by endothelial cell apoptosis and severe loss of microvessels. Lungs from uPAR−/− mice displayed non-specific interstitial pneumonia-like pathological features, both with inflammation and collagen deposition. Pulmonary pathology worsened significantly from 12 to 24 weeks, as shown by a significant increase in alveolar septal width and collagen content. Conclusions uPAR−/− mice are a new animal model closely mimicking the histopathological features of SSc. This model warrants future studies.

Telocytes in minor salivary glands of primary Sjögren's syndrome: association with the extent of inflammation and ectopic lymphoid neogenesis.

It has been recently reported that telocytes, a stromal (interstitial) cell subset involved in the control of local tissue homeostasis, are hampered in the target organs of inflammatory/autoimmune disorders. Since no data concerning telocytes in minor salivary glands (MSGs) are currently available, aim of the study was to evaluate telocyte distribution in MSGs with normal architecture, non‐specific chronic sialadenitis (NSCS) and primary Sjögrens syndrome (pSS)‐focal lymphocytic sialadenitis. Twelve patients with pSS and 16 sicca non‐pSS subjects were enrolled in the study. MSGs were evaluated by haematoxylin and eosin staining and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germinal center (GC)‐like structures. Telocytes were identified by immunoperoxidase‐based immunohistochemistry for CD34 and CD34/platelet‐derived growth factor receptor α double immunofluorescence. Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and encircled both the excretory ducts and the secretory units. In NSCS, despite the presence of a certain degree of inflammation, telocytes were normally represented. Conversely, telocytes were markedly reduced in MSGs from pSS patients compared to normal and NSCS MSGs. Such a decrease was associated with both worsening of glandular inflammation and progression of ectopic lymphoid neogenesis, periductal telocytes being reduced in the presence of smaller inflammatory foci and completely absent in the presence of GC‐like structures. Our findings suggest that a loss of MSG telocytes might have important pathophysiological implications in pSS. The specific pro‐inflammatory cytokine milieu of pSS MSGs might be one of the causes of telocyte loss.

Differential expression of junctional adhesion molecules in different stages of systemic sclerosis.

OBJECTIVE Systemic sclerosis (SSc) is characterized by early perivascular inflammation, microvascular endothelial cell (MVEC) activation/damage, and defective angiogenesis. Junctional adhesion molecules (JAMs) regulate leukocyte recruitment to sites of inflammation and ischemia-reperfusion injury, vascular permeability, and angiogenesis. This study was undertaken to investigate the possible role of JAMs in SSc pathogenesis. METHODS JAM-A and JAM-C expression levels in skin biopsy samples from 25 SSc patients and 15 healthy subjects were investigated by immunohistochemistry and Western blotting. Subcellular localization of JAMs in cultured healthy dermal MVECs and SSc MVECs was assessed by confocal microscopy. Serum levels of soluble JAM-A (sJAM-A) and sJAM-C in 64 SSc patients and 32 healthy subjects were examined by enzyme-linked immunosorbent assay. RESULTS In control skin, constitutive JAM-A expression was observed in MVECs and fibroblasts. In early-stage SSc skin, JAM-A expression was strongly increased in MVECs, fibroblasts, and perivascular inflammatory cells. In late-stage SSc, JAM-A expression was decreased compared with controls. JAM-C was weakly expressed in control and late-stage SSc skin, while it was strongly expressed in MVECs, fibroblasts, and inflammatory cells in early-stage SSc. Surface expression of JAM-A was higher in early-stage SSc MVECs and increased in healthy MVECs stimulated with early-stage SSc sera. JAM-C was cytoplasmic in resting healthy MVECs, while it was recruited to the cell surface upon challenge with early-stage SSc sera. Early-stage SSc MVECs exhibited constitutive surface JAM-C expression. In SSc, increased levels of sJAM-A and sJAM-C correlated with early disease and measures of vascular damage. CONCLUSION Our findings indicate that JAMs may participate in MVEC activation, inflammatory processes, and impaired angiogenesis in different stages of SSc.

Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis.

Objective Systemic sclerosis (SSc) features multiorgan fibrosis orchestrated predominantly by activated myofibroblasts. Endothelial-to-mesenchymal transition (EndoMT) is a transdifferentiation by which endothelial cells (ECs) lose their specific morphology/markers and acquire myofibroblast-like features. Here, we determined the possible contribution of EndoMT to the pathogenesis of dermal fibrosis in SSc and two mouse models. Methods Skin sections were immunostained for endothelial CD31 or vascular endothelial (VE)-cadherin in combination with α-smooth muscle actin (α-SMA) myofibroblast marker. Dermal microvascular ECs (dMVECs) were prepared from SSc and healthy skin (SSc-dMVECs and H-dMVECs). H-dMVECs were treated with transforming growth factor-β1 (TGFβ1) or SSc and healthy sera. Endothelial/mesenchymal markers were assessed by real-time PCR, immunoblotting and immunofluorescence. Cell contractile phenotype was assayed by collagen gel contraction. Results Cells in intermediate stages of EndoMT were identified in dermal vessels of either patients with SSc or bleomycin-induced and urokinase-type plasminogen activator receptor (uPAR)-deficient mouse models. At variance with H-dMVECs, SSc-dMVECs exhibited a spindle-shaped appearance, co-expression of lower levels of CD31 and VE-cadherin with myofibroblast markers (α-SMA+ stress fibres, S100A4 and type I collagen), constitutive nuclear localisation of the EndoMT driver Snail1 and an ability to effectively contract collagen gels. Treatment of H-dMVECs either with SSc sera or TGFβ1 resulted in the acquisition of a myofibroblast-like morphology and contractile phenotype and downregulation of endothelial markers in parallel with the induction of mesenchymal markers. Matrix metalloproteinase-12-dependent uPAR cleavage was implicated in the induction of EndoMT by SSc sera. Conclusions In SSc, EndoMT may be a crucial event linking endothelial dysfunction and development of dermal fibrosis.

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.

Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

IntroductionMicrovascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.MethodsSerum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.ResultsSerum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).ConclusionsOur findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.

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

BackgroundSystemic 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).MethodsWound 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.ResultsTreatment 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.ConclusionsOur 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.