Morphologic evidence of telocytes in human thyroid stromal tissue.

Despite the evidence accumulated over the past decade that telocytes (TCs) are a distinctive, though long neglected, cell entity of the stromal microenvironment of several organs of the human body, to date their localization in the endocrine glands remains almost unexplored. This study was therefore undertaken to examine the presence and characteristics of TCs in normal human thyroid stromal tissue through an integrated morphologic approach featuring light microscopy and ultrastructural analysis. TCs were first identified by immunohistochemistry that revealed the existence of an intricate network of CD34+ stromal cells spread throughout the thyroid interfollicular connective tissue. Double immunofluorescence allowed to clearly differentiate CD34+ stromal cells lacking CD31 immunoreactivity from neighbour CD31+ microvascular structures, and the evidence that these stromal cells coexpressed CD34 and platelet-derived growth factor receptor α further strengthened their identification as TCs. Transmission electron microscopy confirmed the presence of stromal cells ultrastructurally identifiable as TCs projecting their characteristic cytoplasmic processes (i.e., telopodes) into the narrow interstitium between thyroid follicles and blood microvessels, where telopodes intimately surrounded the basement membrane of thyrocytes. Collectively, these morphologic findings provide the first comprehensive demonstration that TCs are main constituents of the human thyroid stroma and lay the necessary groundwork for further in-depth studies aimed at clarifying their putative implications in glandular homeostasis and pathophysiology.

Impairment in the telocyte/CD34+ stromal cell network in human rheumatoid arthritis synovium.

Telocytes (TCs)/CD34+ stromal cells have recently emerged as peculiar interstitial cells detectable in a variety of organs throughout the human body. TCs are typically arranged in networks establishing unique spatial relationships with neighbour cells and likely contributing to the maintenance of tissue homeostasis by both cell-to-cell contacts and releasing extracellular vesicles. Hence, TC defects are being increasingly reported in different pathologies, such as chronic inflammatory and fibrotic conditions. In this regard, TCs/CD34+ stromal cells have been shown to constitute an intricate interstitial network in the subintimal area of the normal human synovial membrane, but whether they are altered in chronic synovitis has yet to be explored. We therefore undertook a morphologic study to compare the distribution of TCs/CD34+ stromal cells between normal synovium and chronically inflamed synovium from patients with rheumatoid arthritis (RA) by using CD34 immunohistochemistry and CD31/CD34 double immunofluorescence. CD34 immunostaining revealed that, at variance with normal synovium, the inflamed and hyperplastic RA synovial tissue was nearly or even completely devoid of TCs/CD34+ stromal cells. Double immunofluorescence confirmed that almost all CD34+ tissue components detectable in RA synovium were blood vessels coexpressing CD31, while a widespread network of CD31- /CD34+ TCs was clearly evident in the whole sublining layer of normal synovium. In the context of the emerging diverse roles of TCs/CD34+ stromal cells in the regulation of tissue homeostasis and structure, the remarkable impairment in their networks herein uncovered in RA synovium may suggest important pathophysiologic implications that will be worth investigating further.

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.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

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.

Angiogenic T cells in primary Sjögren's syndrome: a double-edged sword?

OBJECTIVES: The mechanisms underlying increased cardiovascular risk in primary Sjögren's syndrome (pSS) remain unclear. Since the recently discovered angiogenic T cells (Tang) may participate in endothelial repair by cooperating with endothelial progenitor cells (EPC), we aimed to quantify and characterise Tang in the peripheral blood and minor salivary glands (MSG) of pSS patients. METHODS: Tang (CD3+CD31+CXCR4+) and EPC (CD34+CD133+VEGFR-2+) were quantified by flow cytometry in peripheral blood samples from 36 pSS patients and 20 healthy donors. Tang subsets were assessed on the basis of CD4, CD8 and CD28 expression. Labial MSG sections from 10 pSS patients and 12 non-pSS sicca syndrome controls were subjected to immunofluorescence staining to investigate the presence of Tang and the expression of the CXCR4-ligand stromal cell-derived factor-1 (SDF-1)/CXCL12. RESULTS: Circulating Tang cells were expanded and directly correlated to EPC in pSS. Both Tang and EPC directly correlated with disease activity as calculated with the EULAR Sjögren's syndrome disease activity index (ESSDAI). In pSS, the majority of Tang cells were CD4-CD8- double negative (DN) and lacked CD28 revealing a senescent phenotype. A subset of CD4+, CD8+ and DN Tang cells produced interleukin-17. Immunohistology revealed the exclusive presence of periductal and perivascular infiltrating Tang cells along with increased SDF-1/CXCL12 expression in pSS MSG compared to non-pSS sicca syndrome controls. CONCLUSIONS: In pSS, Tang cells are expanded in peripheral blood and infiltrate MSG. Tang may be novel actors in pSS-related endothelial dysfunction and glandular neo-angiogenesis and inflammation.

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.

Telocyte implications in human pathology: An overview.

Telocytes are a recently described interstitial cell population widely distributed in the stromal compartment of many organs in vertebrates, including humans. Owing to their close spatial relationship with multiple cell types, telocytes are universally considered as 'connecting cells' mostly committed to intercellular signaling by converting the interstitium into an integrated system that drives organ development and contributes to the maintenance of local tissue homeostasis. Increasing evidence indicates that telocytes may cooperate with tissue-resident stem cells to foster organ repair and regeneration, and that telocyte damage and dysfunction may occur in several disorders. The goal of this review is to provide an overview of the most recent findings concerning the implication of telocytes in a variety of pathologic conditions in humans, including heart disease, chronic inflammation and multiorgan fibrosis. Based on recent promising experimental data, there is realistic hope that by targeting telocytes alone or in tandem with stem cells, we might be able to promote organ regeneration and/or prevent irreversible end-stage organ damage in different pathologies.

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.

Telocytes in normal and keratoconic human cornea: an immunohistochemical and transmission electron microscopy study.

Telocytes (TC) are typically defined as cells with telopodes by their ultrastructural features. Their presence was reported in the interstitium of various organs in vertebrates, including humans. However, no study has yet described the presence of TC in the human eye and in particular, within the stromal compartment of the cornea. To address this issue, samples of normal and pathologic (keratoconic) human corneas were tested by immunohistochemistry for CD34, platelet-derived growth factor receptor α (PDGFRα) and c-kit/CD117 or examined by transmission electron microscopy. We found that TC coexpressing CD34 and PDGFRα were distributed throughout the whole normal corneal stroma with different TC subtypes being distinguishable on the basis of the expression of the stemness marker c-kit (i.e. c-kit-positive and c-kit-negative TC subpopulations). Transmission electron microscopy examination confirmed the existence of spindle-shaped and bipolar TC typically displaying two long and thin moniliform telopodes establishing intercellular contacts formed by gap junctions. Keratoconic corneas were characterized by ultrastructural damages and patchy loss of TC with an almost complete depletion of the c-kit-positive TC subpopulation. We propose that TC may contribute to the maintenance of corneal stromal homoeostasis and that, in particular, the c-kit-positive TC subtype might have stemness capacity participating in corneal regeneration and repair processes. Further studies are needed to clarify the differential roles of corneal TC subtypes as well as the possible therapeutic applications of TC in degenerative corneal disorders such as keratoconus.

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-ß1 (TGFß1) 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 TGFß1 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.

Cardiac Telocyte-Derived Exosomes and Their Possible Implications in Cardiovascular Pathophysiology.

Among cardiac interstitial cells, the recently described telocytes (TCs) display the unique ability to build a supportive three-dimensional network formed by their very long and thin prolongations named telopodes. Cardiac TCs are increasingly regarded as pivotal regulators in intercellular signaling with multiple cell types, such as cardiomyocytes, stem/progenitor cells, microvessels, nerve endings, fibroblasts and immune cells, thus converting the cardiac stromal compartment into an integrated system that may drive either heart development or maintenance of cardiac homeostasis in post-natal life. Besides direct intercellular communications between TCs and neighboring cells, different types of TC-released extracellular vesicles (EVs), namely exosomes, ectosomes and multivesicular cargos, may act as shuttles for paracrine molecular signal exchange between cardiac TCs and cardiomyocytes or putative cardiomyocyte progenitors. In this review, we summarize the recent research findings on cardiac TCs and their EVs. We first provide an overview of the general features of TCs, including their peculiar morphological traits and immunophenotypes, intercellular signaling mechanisms and possible functional roles. Thereafter, we describe the distribution of TCs in normal and diseased hearts, as well as their role as intercellular communicators via the release of exosomes and other types of EVs. Finally, the involvement of cardiac TCs in cardiovascular diseases and the potential utility of TC transplantation and TC-derived exosomes in cardiac regeneration and repair are discussed.

Mobilization of lymphatic endothelial precursor cells and lymphatic neovascularization in primary Sjögren's syndrome.

Although lymphatic neovascularization may be a key feature of chronic inflammation, it is almost unexplored in primary Sjögren's syndrome (pSS). A recent study revealed a pro-lymphangiogenic function of interleukin (IL)-17, a leading player in pSS pathogenesis. The aims of the study were to investigate lymphangiogenic mediators and lymphatic vasculature in pSS, as well as their possible association with IL-17. Circulating lymphatic endothelial precursor cells (LEPCs) and Th17 cells were enumerated in pSS patients and healthy donors. VEGF-C and IL-17 levels were assessed in paired serum samples. Lymphatic vasculature, VEGF-C/VEGF receptor (VEGFR)-3 and IL-17 were evaluated in pSS minor salivary glands (MSGs) and compared with normal and non-specific chronic sialadenitis (NSCS) MSGs. Circulating LEPCs were expanded in pSS and correlated with circulating Th17 cells, IL-17 and VEGF-C. In pSS MSGs, a newly formed lymphatic capillary network was found within periductal inflammatory infiltrates and the number of interlobular lymphatic vessels was significantly increased compared with normal and NSCS MSGs. Strong VEGF-C expression was detected in pSS ductal epithelial cells and periductal inflammatory cells. Numerous VEGFR-3(+) infiltrating mononuclear cells were exclusively observed in pSS MSGs. VEGFR-3 expression was strongly increased in lymphatic capillaries of pSS MSGs. IL-17(+) inflammatory cells were preferentially observed around lymphatic vessels in pSS MSGs. This study supports the notion that lymphvasculogenesis and lymphangiogenesis are active in pSS, thereby unmasking a novel aspect of disease pathogenesis. In addition, our results suggest another possible pathogenic role of IL-17 in pSS, further supporting its therapeutic targeting in this disease.

Systemic sclerosis-like histopathological features in the myocardium of uPAR-deficient mice.

OBJECTIVE: Cardiomyopathy is among the leading causes of death from systemic sclerosis (SSc). Urokinase-type plasminogen activator receptor (uPAR)-deficient mice have been recently reported to display important histopathological hallmarks of SSc, including dermal fibrosis, reduced dermal capillary density, and pulmonary fibrosis. Here, we investigated whether uPAR-deficient mice could display the histopathological features of SSc-related cardiomyopathy. METHODS: Ventricular myocardial specimens from uPAR-deficient and wild-type mice at 12 and 24 weeks of age were analysed by both light microscopy and transmission electron microscopy. Picrosirius red staining and hydroxyproline content of myocardial specimens were quantified. Myofibroblast and microvessel counts were determined by immunofluorescence for α-smooth muscle actin and CD31, respectively. Endothelial cell apoptosis was assessed by a combined TUNEL/CD31 immunofluorescence assay. Expression of uPAR in human SSc and control ventricular myocardial autopsy specimens was determined by immunohistochemistry. RESULTS: The myocardium of 24-week-old uPAR-deficient mice displayed focal ischaemic lesions with cardiomyocyte hypertrophy, myofibril rarefaction and contraction band necrosis. At 24 weeks of age, interstitial and perivascular collagen deposition and myofibroblast counts were significantly greater in myocardial tissue of uPAR-deficient mice than in wild-type mice. In uPAR-deficient mice, myocardial fibrosis was paralleled by microvascular endothelial cell apoptosis and reduced capillary density. uPAR expression was significantly downregulated in the myocardium of patients with SSc. CONCLUSIONS: Typical histopathological features of SSc-related cardiomyopathy are mimicked by uPAR-deficient mice. The downregulation of uPAR in the myocardium of patients with SSc may suggest similar underlying pathogenetic mechanisms. uPAR-deficient mice could be used as a preclinical model to study the mechanisms and therapeutic approaches of myocardial involvement in SSc.

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.

Clinical, instrumental, serological and histological findings suggest that hemophilia B may be less severe than hemophilia A.

Recent evidence suggests that patients with severe hemophilia B may have a less severe disease compared to severe hemophilia A. To investigate clinical, radiological, laboratory and histological differences in the arthropathy of severe hemophilia A and hemophilia B, 70 patients with hemophilia A and 35 with hemophilia B with at least one joint bleeding were consecutively enrolled. Joint bleedings (<10, 10-50, >50), regimen of treatment (prophylaxis/on demand), World Federation of Hemophilia, Pettersson and ultrasound scores, serum soluble RANK ligand and osteoprotegerin were assessed in all patients. RANK, RANK ligand and osteoprotegerin expression was evaluated in synovial tissue from 18 hemophilia A and 4 hemophilia B patients. The percentage of patients with either 10-50 or more than 50 hemarthrosis was greater in hemophilia A than in hemophilia B (P<0.001 and P=0.03, respectively), while that with less than 10 hemarthrosis was higher in hemophilia B (P<0.0001). World Federation of Hemophilia (36.6 vs. 20.2; P<0.0001) and ultrasound (10.9 vs. 4.3; P<0.0001) score mean values were significantly higher in hemophilia A patients. Serum osteoprotegerin and soluble RANK ligand were decreased in hemophilia A versus hemophilia B (P<0.0001 and P=0.006, respectively). Osteoprotegerin expression was markedly reduced in synovial tissue from hemophilia A patients. In conclusion, the reduced number of hemarthrosis, the lower World Federation of Hemophilia and ultrasound scores, and higher osteoprotegerin expression in serum and synovial tissue in hemophilia B suggest that hemophilia B is a less severe disease than hemophilia A. Osteoprotegerin reduction seems to play a pivotal role in the progression of arthropathy in hemophilia A.

Telocytes in Chronic Inflammatory and Fibrotic Diseases.

Telocytes are a peculiar stromal (interstitial) cell type implicated in tissue homeostasis and development, as well as in the pathophysiology of several disorders. Severe damage and reduction of telocytes have been reported during fibrotic remodeling of multiple organs in various diseases, including scleroderma, Crohn's disease, ulcerative colitis, and liver fibrosis, as well as in chronic inflammatory lesions like those of primary Sjögren's syndrome and psoriasis. Owing to their close relationship with stem cells, telocytes are also supposed to contribute to tissue repair/regeneration. Indeed, telocytes are universally considered as "connecting cells" mostly oriented to intercellular signaling. On the basis of recent promising experimental findings, in the near future, telocyte transplantation might represent a novel therapeutic opportunity to control the evolution of chronic inflammatory and fibrotic diseases. Notably, there is evidence to support that telocytes could help in preventing abnormal activation of immune cells and fibroblasts, as well as in attenuating the altered matrix organization during the fibrotic process. By targeting telocytes alone or in tandem with stem cells, we might be able to promote regeneration and prevent the evolution to irreversible tissue injury. Besides exogenous transplantation, exploring pharmacological or non-pharmacological methods to enhance the growth and/or survival of telocytes could be an additional therapeutic strategy for many disorders.

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 Masson's 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.

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 Sjögren's 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.

Lymphatic endothelial progenitor cells and vascular endothelial growth factor-C in spondyloarthritis and Crohn's disease: two overlapping diseases?

OBJECTIVES: The role of the lymphatic system in the connection between spondyloarthritis (SpA) and Crohn's disease (CD) remains yet to be elucidated. The aim of the present study was to investigate the circulating levels of lymphatic endothelial progenitor cells (LEPCs) and vascular endothelial growth factor-C (VEGF-C) and their possible correlation with clinical parameters in SpA, SpA associated with CD (SC), and CD. METHODS: Peripheral blood samples from SpA (n=36), SC (n=20) and CD (n=28) patients and 20 age- and sex-matched healthy controls were collected and used for quantification of circulating LEPCs and VEGF-C. LEPCs were identified by fluorescence-activated cell sorting using FITC-CD34, APC-CD133 and PE-VEGFR-3 antibodies. Serum levels of VEGF-C were measured by enzyme-linked immunosorbent assay. The possible correlations between disease duration (< or >10 years; < or >20 years) and clinical activity (BASDAI for SpA or CDAI for CD) and LEPC counts and VEGF-C levels were analysed. RESULTS: Circulating LEPC levels were significantly increased in SpA (p=0.0006) and SC (p=0.0058) patients compared with controls. In CD patients, LEPC counts negatively correlated with disease duration, with lower levels in longstanding disease (>20 years, p=0.018), but were not different from controls. No significant difference in VEGF-C levels was found in SpA, SC and CD compared with controls. Both LEPC and VEGF-C levels were independent of BASDAI and CDAI. CONCLUSIONS: On the basis of our observations, an active mobilisation of lymphatic endothelial cell precursors was observed only for spondylitis involvement.