The contribution of endothelial cells to tissue fibrosis.

PURPOSE OF REVIEW: Tissue fibrosis is an increasingly prevalent condition associated with various diseases and heavily impacting on global morbidity and mortality rates. Growing evidence indicates that common cellular and molecular mechanisms may drive fibrosis of diverse cause and affecting different organs. The scope of this review is to highlight recent findings in support for an important role of vascular endothelial cells in the pathogenesis of fibrosis, with a special focus on systemic sclerosis as a prototypic multisystem fibrotic disorder. RECENT FINDINGS: Although transition of fibroblasts to chronically activated myofibroblasts is widely considered the central profibrotic switch, the endothelial cell involvement in development and progression of fibrosis has been increasingly recognized over the last few years. Endothelial cells can contribute to the fibrotic process either directly by acting as source of myofibroblasts through endothelial-to-myofibroblast transition (EndMT) and concomitant microvascular rarefaction, or indirectly by becoming senescent and/or secreting a variety of profibrotic and proinflammatory mediators with consequent fibroblast activation and recruitment of inflammatory/immune cells that further promote fibrosis. SUMMARY: An in-depth understanding of the mechanisms underlying EndMT or the acquisition of a profibrotic secretory phenotype by endothelial cells will provide the rationale for novel endothelial cell reprogramming-based therapeutic approaches to prevent and/or treat fibrosis.

The differential crosstalk of the skin-gut microbiome axis as a new emerging actor in systemic sclerosis.

OBJECTIVES: We characterized the microbiota in SSc, focusing on the skin-oral-gut axis and the serum and faecal free fatty acid (FFA) profile. METHODS: Twenty-five SSc patients with ACA or anti-Scl70 autoantibodies were enrolled. The microbiota of faecal, saliva and superficial epidermal samples was assessed through next-generation sequencing analysis. GC-MS was used to quantify faecal and serum FFAs. Gastrointestinal symptoms were investigated with the University of California Los Angeles Scleroderma Clinical Trial Consortium Gastrointestinal Tract Instrument (UCLA GIT-2.0) questionnaire. RESULTS: The ACA+ and anti-Scl70+ groups displayed different cutaneous and faecal microbiota profiles. The classes of cutaneous Sphingobacteriia and Alphaproteobacteria, the faecal phylum Lentisphaerae, the levels of the classes Lentisphaeria and Opitutae, and the genus NA-Acidaminococcaceae were significantly higher in faecal samples from the ACA+ patients than in samples from the anti-Scl70+ patients. The cutaneous Sphingobacteria and the faecal Lentisphaerae were significantly correlated (rho = 0.42; P = 0.03). A significant increase in faecal propionic acid was observed in ACA+ patients. Moreover, all levels of faecal medium-chain FFAs and hexanoic acids were significantly higher in the ACA+ group than in the anti-Scl70+ group (P < 0.05 and P < 0.001, respectively). In the ACA+ group, the analysis of the serum FFA levels showed an increasing trend in valeric acid. CONCLUSION: Different microbiota signatures and FFA profiles were found for the two groups of patients. Despite being in different body districts, the cutaneous Sphingobacteria and faecal Lentisphaerae appear interdependent.

Soluble guanylate cyclase stimulation fosters angiogenesis and blunts myofibroblast-like features of systemic sclerosis endothelial cells.

OBJECTIVES: In SSc, angiogenesis impairment advances in parallel with the development of fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-mesenchymal transition (EndoMT). Soluble guanylate cyclase (sGC) stimulation has shown antifibrotic effects in SSc skin fibroblasts and mouse models. Here, we investigated the effects of pharmacological sGC stimulation on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-MVECs). METHODS: To determine whether sGC stimulation affected cell viability/proliferation, SSc-MVECs and healthy dermal MVECs (H-MVECs) were challenged with the sGC stimulator (sGCS) MK-2947 and assayed by annexin V/propidium iodide flow cytometry and the water-soluble tetrazolium salt (WST-1) assay. To study angiogenesis and EndoMT, MK-2947-treated SSc-MVECs were subjected to wound healing and capillary morphogenesis assays and analysed for the expression of endothelial/myofibroblast markers and contractile ability. RESULTS: MK-2947 treatment did not affect H-MVEC viability/proliferation, while it significantly increased SSc-MVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-MVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in the expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 mesenchymal markers was also found in MK-2947-treated SSc-MVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-MVECs to contract collagen gels and reduced phosphorylated-extracellular signal-regulated kinases 1 and 2 protein levels. CONCLUSION: These findings demonstrate for the first time that pharmacological sGC stimulation effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-MVECs, thus providing new evidence for repurposing sGCSs for SSc.

Current Trends in Vascular Biomarkers for Systemic Sclerosis: A Narrative Review.

Systemic sclerosis (SSc, scleroderma) is a multifaceted rare connective tissue disease whose pathogenesis is dominated by immune dysregulation, small vessel vasculopathy, impaired angiogenesis, and both cutaneous and visceral fibrosis. Microvascular impairment represents the initial event of the disease, preceding fibrosis by months or years and accounting for the main disabling and/or life-threatening clinical manifestations, including telangiectasias, pitting scars, periungual microvascular abnormalities (e.g., giant capillaries, hemorrhages, avascular areas, ramified/bushy capillaries) clinically detectable by nailfold videocapillaroscopy, ischemic digital ulcers, pulmonary arterial hypertension, and scleroderma renal crisis. Despite a variety of available treatment options, treatment of SSc-related vascular disease remains problematic, even considering SSc etherogenity and the quite narrow therapeutic window. In this context, plenty of studies have highlighted the great usefulness in clinical practice of vascular biomarkers allowing clinicians to assess the evolution of the pathological process affecting the vessels, as well as to predict the prognosis and the response to therapy. The current narrative review provides an up-to-date overview of the main candidate vascular biomarkers that have been proposed for SSc, focusing on their main reported associations with characteristic clinical vascular features of the disease.

A candidate gene study reveals association between a variant of the SRp55 splicing factor gene and systemic sclerosis.

OBJECTIVES: To examine the possible implication of the mRNA-binding protein serine/arginine protein 55 (SRp55, also known as SRSF6) rs2235611 single nucleotide polymorphism (SNP) in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotype. METHODS: A total population of 872 white Italian individuals (414 SSc patients, 458 controls) was studied. SSc patients were assessed for limited and diffuse cutaneous subsets and the presence of autoantibodies, interstitial lung disease (ILD), and nailfold videocapillaroscopy (NVC) abnormalities. The SRp55 rs2235611 SNP was genotyped by TaqMan real-time PCR. RESULTS: SRp55 rs2235611 genotype distribution and allele frequency were similar in SSc and healthy controls, though a trend toward significance was observed for genotype distribution (p=0.07). The SRp55 rs2235611 AA genotype significantly influenced the predisposition to SSc (p= 0.03). The SRp55 rs2235611 A minor allele and AA genotype showed a significant risk association with susceptibility to SSc-related ILD (A allele: p=0.046; AA genotype: p=0.007). A significant association of the AA genotype with SSc late NVC pattern was also found (p=0.006). After Bonferroni correction for multiple comparisons, the risk association of the SRp55 rs2235611 AA genotype with SSc-related ILD and late NVC pattern remained significant (padj=0.049 and padj=0.042, respectively). CONCLUSIONS: The SRp55 rs2235611 AA genotype significantly influences the susceptibility to SSc, and specifically associates with the presence of SSc-related ILD and late NVC pattern. Further in-depth studies on the SRp55 gene locus will hopefully contribute to extend our knowledge of the genetic predisposition to major SSc-related manifestations such as pulmonary fibrosis and peripheral microvasculopathy.

The Soluble Guanylate Cyclase Stimulator BAY 41-2272 Attenuates Transforming Growth Factor ß1-Induced Myofibroblast Differentiation of Human Corneal Keratocytes.

Corneal transparency, necessary for vision and depending on the high organization of stromal extracellular matrix, is maintained by keratocytes. Severe or continuous corneal injuries determine exaggerated healing responses resulting in the formation of irreversible fibrotic scars and vision impairment. Soluble guanylate cyclase (sGC) stimulation demonstrated antifibrotic effects in both experimental fibrosis and human lung and skin fibroblasts. Here, we assessed whether sGC stimulation with BAY 41-2272 could attenuate transforming growth factor ß1 (TGFß1)-induced myofibroblast differentiation of human corneal keratocytes. Cells were challenged with TGFß1, with/without BAY 41-2272 preincubation, and subsequently assessed for viability, proliferation, migration, chemoinvasion, as well for the expression of myofibroblast/fibroblast activation markers and contractile abilities. Treatment with BAY 41-2272 did not affect keratocyte viability, while preincubation of cells with the sGC stimulator was able to inhibit TGFß1-induced proliferation, wound healing capacity, and invasiveness. BAY 41-2272 was also able to attenuate TGFß1-induced myofibroblast-like profibrotic phenotype of keratocytes, as demonstrated by the significant decrease in ACTA2, COL1A1, COL1A2, FN1 and PDPN gene expression, as well as in α-smooth muscle actin, α-1 chain of type I collagen, podoplanin, vimentin and N-cadherin protein expression. Finally, BAY 41-2272 significantly counteracted the TGFß1-induced myofibroblast-like ability of keratocytes to contract collagen gels, reduced phosphorylated Smad3 protein levels, and attenuated gene expression of proinflammatory cytokines. Collectively, our data show for the first time that BAY 41-2272 is effective in counteracting keratocyte-to-myofibroblast transition, thus providing the rationale for the development of sGC stimulators as novel promising modulators of corneal scarring and fibrosis.

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31-/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

A Two-Step Immunomagnetic Microbead-Based Method for the Isolation of Human Primary Skin Telocytes/CD34+ Stromal Cells.

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31-/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα-/vimentin+) and fibroblasts (CD31-/CD34-/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.

A new avenue in the pathogenesis of systemic sclerosis: the molecular interface between the endothelial and the nervous systems.

Systemic sclerosis (SSc) is a connective tissue disorder characterised by immune dysregulation, endothelial cell dysfunction followed by defective vascular repair and neovascularization and progressive tissue fibrosis of the skin and internal organs, whose pathophysiology remains to be fully elucidated. Perturbed neuroendothelial control mechanisms comprising either endothelial cell or peripheral nerve fiber impairment are supposed to play an important role in the onset of Raynaud's phenomenon and development of microvascular abnormalities which are the earliest events and key features of SSc. Such pathogenic neuroendothelial mechanisms may trigger both the early endothelial cell damage and the subsequent loss of peripheral microvascular integrity characterised by the lack of compensatory angiogenesis. Of note, the vascular and nervous systems have several anatomical similarities that extend to molecular level, and the molecular mechanisms of nerve regulation are shared by the vascular system. In this context, increasing evidence demonstrated that endothelial cells express receptors for axon guidance molecules, including Ephrin family receptor tyrosine kinases, Neuropilins, Plexins, Robos, and UNC5B that are able to respond to their soluble neuroendothelial trophic ligands, such as Semaphorins and Slits, to guide the sprouting of endothelial tip cells. Here, we first provide a historical view of neuroendothelial control mechanism alterations in the pathogenesis of SSc, and then discuss the emerging role of a class of molecules sharing neurogenic and angiogenic properties, such as members of Semaphorin/Plexin/Neuropilin and Slit/Roundabout families, in SSc-related peripheral microvasculopathy.

Systemic Sclerosis Serum Significantly Impairs the Multi-Step Lymphangiogenic Process: in Vitro Evidence.

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.

Slit2/Robo4 axis may contribute to endothelial cell dysfunction and angiogenesis disturbance in systemic sclerosis.

OBJECTIVE: In systemic sclerosis (SSc), early microvascular injury is followed by impaired angiogenesis and peripheral capillary loss. Here, we investigated the possible contribution of the neurovascular guidance molecule Slit2 and its Roundabout (Robo) receptors to SSc-related endothelial cell dysfunction. METHODS: Circulating Slit2 levels were measured in patients with SSc and healthy controls. Slit2, Robo1 and Robo4 expression was investigated in SSc and healthy skin biopsies and explanted dermal microvascular endothelial cells (MVECs). Slit2/Robo4 function in MVEC angiogenesis was studied by cell viability, wound healing and capillary-like tube formation assays. RESULTS: Circulating Slit2 was significantly increased in either SSc or patients with a very early diagnosis of SSc (VEDOSS) compared with controls. Interestingly, serum Slit2 levels were raised in patients with VEDOSS with nailfold videocapillaroscopy (NVC) abnormalities, while they were similar in VEDOSS with normal NVC and controls. In SSc, Slit2 and Robo4 expression was upregulated in clinically affected skin and explanted MVECs in respect to controls. The angiogenic performance of healthy MVECs was significantly reduced after challenge with recombinant human Slit2 or SSc sera. These inhibitory effects were significantly attenuated when SSc sera were preincubated with an anti-Slit2 blocking antibody. In vitro angiogenesis was severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or ROBO4 gene silencing. Slit2/Robo4 axis interfered with angiogenesis through the inhibition of Src kinase phosphorylation. CONCLUSIONS: In SSc, increased circulating levels of Slit2 and activation of the Slit2/Robo4 antiangiogenic axis may contribute to peripheral microangiopathy since the very early phase of the disease.

Recent Insights into Cellular and Molecular Mechanisms of Defective Angiogenesis in Systemic Sclerosis.

In systemic sclerosis (SSc, or scleroderma), defective angiogenesis, clinically manifesting with abnormal capillary architecture and severe capillary reduction, represents a hallmark of early-stage disease, usually preceding the onset of tissue fibrosis, and is caused by several cellular and molecular mechanisms affecting microvascular endothelial cells with different outcomes. Indeed, once damaged, endothelial cells can be dysfunctionally activated, thus becoming unable to undergo angiogenesis and promoting perivascular inflammation. They can also undergo apoptosis, transdifferentiate into profibrotic myofibroblasts, or acquire a senescence-associated secretory phenotype characterized by the release of exosomes and several profibrotic and proinflammatory mediators. In this narrative review, we aimed to give a comprehensive overview of recent studies dealing with the cellular and molecular mechanisms underlying SSc defective angiogenesis and the related endothelial cell dysfunctions, mainly the endothelial-to-mesenchymal transition process. We also discussed potential novel vascular treatment strategies able to restore the angiogenic process and reduce the endothelial-to-mesenchymal transition in this complex disease.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor ß (TGFß)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFß1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFß1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFß1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFß1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1ß and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

Blockade of Sialylation with Decrease in Polysialic Acid Levels Counteracts Transforming Growth Factor ß1-Induced Skin Fibroblast-to-Myofibroblast Transition.

Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor ß1 (TGFß1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFß1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFß1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFß1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFß1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis.

Lymphatic Endothelial-to-Myofibroblast Transition: A Potential New Mechanism Underlying Skin Fibrosis in Systemic Sclerosis.

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-ß1 (TGFß1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFß1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development.

Extracellular Lactic Acidosis of the Tumor Microenvironment Drives Adipocyte-to-Myofibroblast Transition Fueling the Generation of Cancer-Associated Fibroblasts.

Lactic acidosis characterizes the tumor microenvironment (TME) and is involved in the mechanisms leading to cancer progression and dissemination through the reprogramming of tumor and local host cells (e.g., endothelial cells, fibroblasts, and immune cells). Adipose tissue also represents a crucial component of the TME which is receiving increasing attention due to its pro-tumoral activity, however, to date, it is not known whether it could be affected by the acidic TME. Now, emerging evidence from chronic inflammatory and fibrotic diseases underlines that adipocytes may give rise to pathogenic myofibroblast-like cells through the adipocyte-to-myofibroblast transition (AMT). Thus, our study aimed to investigate whether extracellular acidosis could affect the AMT process, sustaining the acquisition by adipocytes of a cancer-associated fibroblast (CAF)-like phenotype with a pro-tumoral activity. To this purpose, human subcutaneous adipose-derived stem cells committed to adipocytes (acADSCs) were cultured under basal (pH 7.4) or lactic acidic (pH 6.7, 10 mM lactate) conditions, and AMT was evaluated with quantitative PCR, immunoblotting, and immunofluorescence analyses. We observed that lactic acidosis significantly impaired the expression of adipocytic markers while inducing myofibroblastic, pro-fibrotic, and pro-inflammatory phenotypes in acADSCs, which are characteristic of AMT reprogramming. Interestingly, the conditioned medium of lactic acidosis-exposed acADSC cultures was able to induce myofibroblastic activation in normal fibroblasts and sustain the proliferation, migration, invasion, and therapy resistance of breast cancer cells in vitro. This study reveals a previously unrecognized relationship between lactic acidosis and the generation of a new CAF-like cell subpopulation from adipocytic precursor cells sustaining tumor malignancy.

Immunohistochemical and ultrastructural identification of telocytes in the lamina propria of human vaginal mucosa.

Since their relatively recent discovery, telocytes (TCs) have been described as peculiar cells strategically positioned in the stromal tissue component of multiple organ systems of the mammalian body including female reproductive organs (i.e., ovary, uterine tube, and uterus). Nevertheless, current knowledge of TCs in the vagina is very limited. The present study was therefore undertaken to investigate the existence and characteristics of TCs in the stromal tissue of human vaginal mucosa by means of immunohistochemistry, immunofluorescence confocal microscopy, and transmission electron microscopy. In the vaginal lamina propria, TCs were first identified by CD34 immunohistochemistry that revealed the presence of CD34+ stromal cells arranged in networks, especially around blood vessels. Double immunofluorescence confocal microscopy allowed to precisely distinguish the perivascular networks of CD34+ stromal cells lacking CD31 immunoreactivity from adjacent CD31+ microvessels. All the perivascular networks of TCs/CD34+ stromal cells situated in the vaginal lamina propria coexpressed platelet-derived growth factor receptor α, which strengthened their identification as TCs. Instead, vaginal mucosal TCs were immunophenotypically negative for c-kit/CD117. The ultrastructural examination confirmed the presence of TCs, namely stromal cells with characteristic cytoplasmic processes (i.e., telopodes) forming labyrinthine networks around blood vessels and releasing extracellular vesicles. Together, our morphological findings provide the first comprehensive demonstration that TCs reside in the human vaginal lamina propria, thus paving the way for further investigation of their putative functions in vaginal mucosal homeostasis and pathophysiology.

Circulating Neurovascular Guidance Molecules and Their Relationship with Peripheral Microvascular Impairment in Systemic Sclerosis.

Systemic sclerosis (SSc, scleroderma) is a complex connective tissue disease whose earliest clinical manifestations are microvascular tone dysregulation and peripheral microcirculatory abnormalities. Following previous evidence of an association between circulating neurovascular guidance molecules and SSc disturbed angiogenesis, here, we measured the levels of soluble neuropilin 1 (sNRP1), semaphorin 3E (Sema3E), and Slit2 by enzyme-linked immunosorbent assay in serum samples from a large case series of 166 SSc patients vs. 110 healthy controls. We focused on their possible correlation with vascular disease clinical features and applied logistic regression analysis to determine which of them could better reflect disease activity and severity. Our results demonstrate that, in SSc: (i) sNRP1 is significantly decreased, with lower sNRP1 serum levels correlating with the severity of nailfold videocapillaroscopy (NVC) abnormalities and the presence of ischemic digital ulcers (DUs); (ii) both Sema3E and Slit2 are increased, with Sema3E better reflecting early NVC abnormalities; and (iii) higher Sema3E correlates with the absence of DUs, while augmented Slit2 associates with the presence of DUs. Receiver operator characteristics curve analysis revealed that both circulating sNRP1 and Sema3E show a moderate diagnostic accuracy. Moreover, logistic regression analysis allowed to identify sNRP1 and Sema3E as more suitable independent biomarkers reflecting the activity and severity of SSc-related peripheral microvasculopathy.

Decreased Serum Levels of SIRT1 and SIRT3 Correlate with Severity of Skin and Lung Fibrosis and Peripheral Microvasculopathy in Systemic Sclerosis.

Systemic sclerosis (SSc, scleroderma) is a severe autoimmune connective tissue disease characterized by widespread peripheral microvasculopathy, and progressive cutaneous and visceral fibrosis, leading to significant organ dysfunction. Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases with pleiotropic effects on a variety of biological processes, including metabolism, cell survival, and aging. In the last decades, increasing studies have explored the contribution of SIRTs to the pathogenesis of SSc, highlighting a significant antifibrotic effect of both SIRT1 and SIRT3. On these bases, the aim of this study was to measure circulating SIRT1 and SIRT3 levels by enzyme-linked immune-sorbent assay in a well-characterized cohort of SSc patients (n = 80) and healthy controls (n = 71), focusing on their possible association with disease clinical features, and their potential as biomarkers reflecting SSc activity and severity. Significantly decreased serum levels of both SIRT1 and SIRT3 were found in SSc patients compared to controls. In SSc, the reduction in circulating SIRT1 and SIRT3 associated with a greater extent of cutaneous fibrosis, presence of interstitial lung disease, and worse pulmonary function. Serum SIRT1 and SIRT3 decrease also correlated with the severity of nailfold microvascular damage, with SIRT3 levels being additionally related to the occurrence of digital ulcers. The levels of these two proteins showed a direct correlation with one another in the circulation of SSc patients. Of the two SIRTs, serum SIRT3 was found to better reflect disease activity and severity in a logistic regression analysis model. Our findings suggest that serum SIRT1 and SIRT3 may represent novel potential biomarkers of increased risk for a more severe, life-threatening SSc disease course.

The Role of Pro-fibrotic Myofibroblasts in Systemic Sclerosis: From Origin to Therapeutic Targeting.

Systemic sclerosis (SSc, scleroderma) is a complex connective tissue disorder characterized by multisystem clinical manifestations resulting from immune dysregulation/autoimmunity, vasculopathy, and, most notably, progressive fibrosis of the skin and internal organs. In recent years, it has been observed that the main drivers of SSc-related tissue fibrosis are myofibroblasts, a type of mesenchymal cells with both the extracellular matrix-synthesizing features of fibroblasts and the cytoskeletal characteristics of contractile smooth muscle cells. The accumulation and persistent activation of pro-fibrotic myofibroblasts during SSc development and progression result in elevated mechanical stress and reduced matrix plasticity within the affected tissues and may be ascribed to a reduced susceptibility of these cells to pro-apoptotic stimuli, as well as their increased formation from tissue-resident fibroblasts or transition from different cell types. Given the crucial role of myofibroblasts in SSc pathogenesis, finding the way to inhibit myofibroblast differentiation and accumulation by targeting their formation, function, and survival may represent an effective approach to hamper the fibrotic process or even halt or reverse established fibrosis. In this review, we discuss the role of myofibroblasts in SSc-related fibrosis, with a special focus on their cellular origin and the signaling pathways implicated in their formation and persistent activation. Furthermore, we provide an overview of potential therapeutic strategies targeting myofibroblasts that may be able to counteract fibrosis in this pathological condition.