Therapeutic potential of human-induced pluripotent stem cell-derived endothelial cells in a bleomycin-induced scleroderma mouse model

Vascular injury and destruction of endothelial cells (ECs) are the early events in scleroderma (SSc) patients. This study aims to investigate the therapeutic potential of human-induced pluripotent stem cell-derived ECs (hiPSC-ECs) to treat SSc. We have assessed the functional differentiation of hiPSC-ECs and compared them with human embryonic stem cell-derived ECs (hESC-ECs) by a variety of in vitro experimental approaches. Additionally, we evaluated the therapeutic potential of hiPSC-ECs in a bleomycin-induced SSc mouse model. Our results demonstrated that hiPSC-ECs and hESC-ECs showed similar maximum expressions of FLK1 (early EC marker) at day five during differentiation. After sorting and culturing, the FLK1-positive cells exhibited spindle and subsequent endothelial cobblestone morphology in EGM2 medium. The hESC-ECs and hiPSC-ECs also expressed late EC markers CD31 (68% and 75%), CD144 (50% and 61%), CD146 (46% and 61%), and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) uptake (55% and 63%), respectively. They additionally formed capillary-like structures on Matrigel. Analyses of the transplantation of sorted CD31-positive hiPSC-ECs into the bleomycin-induced SSc mouse model demonstrated that these cells participate in recovery of the damaged vessels. There was a reduction in collagen content; the number of total and degranulated mast cells returned to their normal state, and bleomycin-induced wounds as well as skin fibrosis improved four weeks after transplantation of hiPSC-ECs. Our findings have shown that the differentiation process from hESCs and hiPSCs to vascular cell components is similar. Additionally, this is the first study to determine the therapeutic potential of vascular cells from hiPSCs in the treatment of an SSc model. In the future, with further validation, these may be used as an appropriate source for the treatment of SSc patients.     

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 mug of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-beta and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-beta1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-beta mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.     

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 μg of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-β and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4⁺ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4⁺ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-β mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.     

Mast cells in the mouse mammary gland--correlation with the development of lactiferous structures

Specimens of mouse mammary glands obtained from animals being in different phases of reproductive cycle were collected. After staining MCN, the total mast cell area (TMC), mean mast cell area (MMC), and lactiferous structure density index (LDI) were examined in sections, using a computer image analysis system. Statistically significant (P < 0.05) results were: 1. An increase in MCN observed in Group I (4-5 and 9-10 days of pregnancy), a decrease in MCN observed in Group II (2nd and 10th day of lactation); 2. Changes of TMC fully corresponding to changes of MCN; 3. Increase in MMC observed in Group I at mid (days 9-10) and at the end (days 18-19) of pregnancy, decrease in MMC observed in Group IIB (10th day of lactation); 4. LDI (%) higher at the end of pregnancy (Group IC) and during lactation (groups IIA, IIB), compared with control (23.5 +/- 4.12, 37.6 +/- 3.24, 71.0 +/- 4.33 vs. 3.8 +/- 0.39). The observed changes in the number and size of MC strictly correspond to physiological phenomena leading to alternation of the mouse mammary gland functional status by development/involution of the lactiferous structures.     

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

Introduction  

Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.     

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.     

Delayed-type hypersensitivity to elastase-soluble lung peptides in the tight-skin (Tsk) mouse

The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responses when challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy 1.2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse.     

Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice

Mast cells mediate allergies, hypersensitivities, host defense, and venom neutralization. An area of recent interest is the contribution of mast cells to inflammatory pain. Here we found that specific, local activation of mast cells produced plantar hyperalgesia in mice. Basic secretagogue compound 48/80 induced plantar mast cell degranulation accompanied by thermal hyperalgesia, tissue edema, and neutrophil influx in the hindpaws of ND4 Swiss mice. Blocking mast cell degranulation, neutrophil extravasation, and histamine signaling abrogated these responses. Compound 48/80 also produced edema, pain, and neutrophil influx in WT C57BL/6 but not in genetically mast cell-deficient C57BL/6-Kit(W-sh)(/)(W-sh) mice. These responses were restored following plantar reconstitution with bone marrow-derived cultured mast cells.     

Mast cells and inflammation

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Mast cells and hemangioma

Hemangioma is a primary tumor of the microvasculature in which angiogenesis is initially excessive, followed by spontaneous regression of the newly formed vessels, with the cellular parenchyma gradually being replaced with fibrofatty tissue. Mast cells, which are highly heterogenous in terms of their morphology, function, and metabolic products, have been implicated in the pathophysiology of hemangioma. Csaba stain shows that mast cells are predominantly of the biogenic amine phenotype throughout the development of hemangioma. The predominance of this phenotype remains unaltered following successful steroid therapy, although their number increases fourfold. Mast cells, all of which stain positive for tryptase, and those that stain positive for chymase as well, have been identified in hemangioma biopsy specimens throughout the three developmental phases. The total number of mast cells is highest during the involuting phase, less in the involuted phase, and least in the proliferative phase. The proportion of mast cells that contain both tryptase and chymase decreases from the proliferative through involuting to the involuted phase. This decreasing proportion of mast cells that contain both tryptase and chymase with ongoing involution parallels that of progressive deposition of the extracellular matrix as indicated by increasing fibrosis and fatty deposition. The short-chain type VIII collagen, thought to play a key role in angiogenesis, has been detected throughout the developmental phases of hemangioma. It has been postulated that this collagen, which is produced early in new vessel development, provides a substratum to facilitate the migration of endothelial cells. It may also facilitate the deposition of other extracellular constituents and influence cell movement and the maintenance of cell phenotypes. The intracellular localization of type VIII collagen in mast cells only in the early proliferative phase suggests that there is an active synthesis by mast cells during this phase. The increasing extracellular localization during hemangioma development may be caused by an increased secretion of protein from intracellular stores. The increased number of mast cells during the involuting phase indicates that these cells may play a role in the regression of hemangioma. This is in contrast to the large body of evidence showing the proangiogenic role of mast cells. The proportion of proliferating mast cells decreases, whereas the proportion of mast cells positive for clusterin/apolipoprotein J increases with ongoing involution of hemangioma. Clusterin/apolipoprotein J expression has been considered as a prominent marker of apoptotic cell loss. The presence of clusterin/apolipoprotein J granules both in the adjacent endothelial cells and in capillary lumens suggests that mast cells may be secreting this apoptotic modulator to promote the regression of hemangioma. Certain effectors produced by mast cells may participate in the development of hemangioma. It has been proposed that one of the functions of mast cells is to release factors leading to the regression of hemangioma. The evidence suggests that although mast cells may have a function in the endothelial proliferation in hemangioma, they also play a crucial role in the regression of this tumor. However, the roles of mast cells in the life cycle of hemangioma are likely to be complex and may involve stimulators of angiogenesis in the proliferative phase but inhibitors in later phases.     

Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis

We explored the pathologic changes in the skin of mice undergoing a chronic graft-versus-host (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin-a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2----BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21-63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis.     

Mast cells in the human brain

Mast cells, as adjudged by the metachromatic staining of their cytoplasmic granules, were found in 79% of the 97 humans brains studied. They were most numerous and most consistently present in the infundibulum, pineal organ, area postrema and choroid plexuses. They were also numerous in the leptomeninges surrounmding the pineal organ and infundibulum. Occasional mast cells were also seen within the supraoptic crest, the subfornical organ, the ventricles and the leptomeninges at sites other than over the infundibulum and pineal organ. They were not detectable elsewhere in the brain or spinal cord. In the infundibulum, pineal organ, area postrema and telencephalic choroid plexuses mast cells were most numerous in young individuals (i.e., 0-19 years of age); thereafter, their numbers progressively decreased with aging. Elsewhere mast cell numbers remained about the same with aging. Except in the area postrema where mast cells were more numerous and more consistently present in males, sex-related differences in mast cell number or distribution were not detected. No differences in either the abundance, the distribution or the percentage of individuals possessing mast cells at any of these sites were apparent between 'normative' brains, lesioned brains ('stroke', lobotomy, etc.) or those from individuals with either congenital or acquired encephalopathies.     

Mast cells in a murine lung ischemia-reperfusion model of primary graft dysfunction

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit^+/+) and mast cell-deficient (Kit^W-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient Kit^W-sh/W-sh mice did not differ from those of Kit^+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected Kit^W-sh/W-sh lungs. However, lung injury in BMCMC-injected Kit^W-sh/W-sh and Kit^+/+ mice did not differ in globally mast cell-deficient, uninjected Kit^W-sh/W-sh mice or in wild-type Kit^+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

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