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Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, α5β1 integrin, and αvβ3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.

All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.

BackgroundIdiopathic pulmonary fibrosis (IPF) is characterised by damage to the epithelial layer, closely associated with the alveolar basement membrane (BM). We aimed to investigate how type IV collagen (COL4) in the BM changes with the progression of IPF.MethodsCOL4 synthesis (PRO-C4) was detected in blood by the nordicPRO-C4 biomarker in patients with IPF from the two prospective, multicentre, observational, longitudinal cohorts, pulmonary fibrosis biomarker (PFBIO) and prospective observation of fibrosis in the lung clinical endpoints (PROFILE). PRO-C4 trajectories over 12 months were compared between progressors and non-progressors by linear mixed effects regression models. Rate of change in PRO-C4 and lung function were compared by Bayesian bivariate longitudinal models. Cox proportional hazards models analysed baseline PRO-C4 and 3 years mortality. COL4 staining in IPF and non-IPF lungs was evaluated by immunohistochemistry.ResultsIn PFBIO and PROFILE, 51/220 (23.2%) and 221/459 (48.1%) patients, respectively, had progressive disease at 12 months. Longitudinal PRO-C4 levels were higher in progressors versus non-progressors (average differences: PFBIO 21.5% (95% CI 3.4% to 42.9%, p=0.0184); PROFILE 10.9% (95% CI 0.8% to 22.1%; p=0.0340). Monthly rate of change in PRO-C4 was steeper in non-survivors versus survivors (mean difference up to 3.12% (95% CI 0.35% to 5.91%)) and was inversely correlated with the change in lung function. High baseline PRO-C4 was associated with increased mortality risk in PFBIO (HR 2.55 (95% CI 1.27 to 5.12), p=0.0083). COL4 staining was higher in IPF versus non-IPF lung but was less obvious in end-stage tissue.ConclusionsHigh and increasing serological PRO-C4 levels were prognostic for progression in two independent IPF cohorts. This study suggests that COL4 synthesis assessed by PRO-C4 is a pathologically relevant biomarker of alveolar BM repair in IPF.

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Defects in type IV collagen, a collagenous protein involved in the formation of basement membranes, have been implicated in hereditary Alport's syndrome and acquired Goodpasture's syndrome. Mutations in genes corresponding to the building blocks of type IV collagen cause Alport's syndrome, whereas autoantibodies against structures that are usually hidden in the recesses of collagen IV cause Goodpasture's syndrome. Basement membranes form a complex surface on which epithelial cells reside. These membranes provide morphogenic cues that determine the fate of cells, the polarization of subcellular constituents, and the location of cell receptors and transporters. 1 – 3 Basement membranes are assembled through an interweaving of type IV collagen (collagen IV) with laminins, nidogen, and sulfated proteoglycans. 4 , 5 Collagen IV belongs to a family of collagenous proteins that has at least 25 distinct members. The COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, and COL4A6 genes 6 – 13 encoding the six chains of collagen IV — α1(IV) through α6(IV) — are selectively expressed in different membranes . . .

Pulmonary arterial hypertension (PAH) is a progressive disease which causes right ventricular (RV) failure. Canstatin, a C-terminal fragment of type IV collagen α2 chain, is expressed in various rat organs. However, the expression level of canstatin in plasma and organs during PAH is still unclear. We aimed to clarify it and further investigated the protective effects of canstatin in a rat model of monocrotaline-induced PAH. Cardiac functions were assessed by echocardiography. Expression levels of canstatin in plasma and organs were evaluated by enzyme-linked immunosorbent assay and Western blotting, respectively. PAH was evaluated by catheterization. RV remodeling was evaluated by histological analyses. Real-time polymerase chain reaction was performed to evaluate RV remodeling-related genes. The plasma concentration of canstatin in PAH rats was decreased, which was correlated with a reduction in acceleration time/ejection time ratio and an increase in RV weight/body weight ratio. The protein expression of canstatin in RV, lung and kidney was decreased in PAH rats. While recombinant canstatin had no effect on PAH, it significantly improved RV remodeling, including hypertrophy and fibrosis, and prevented the increase in RV remodeling-related genes. We demonstrated that plasma canstatin is decreased in PAH rats and that administration of canstatin exerts cardioprotective effects.

Abstract Aims Collagen-derived peptides such as collagen I C-terminal telopeptide (CITP) and procollagen III N-terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression. Methods and results In 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = −0.41, P < 0.001 and r = −0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B-type natriuretic peptide (r = 0.32, P = 0.003) and γ-glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end-diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end-diastolic volume index nor with ejection fraction. Each collagen-derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA. Conclusions Our study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra-cardiac organ injury in heart failure.

Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1–20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10–20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

A known consequence of the large weight loss after bariatric surgery is the appearance of large skinfolds, particularly in the abdomen region of the patients. The balance between the synthesis of extracellular matrix (ECM) components and their proteolysis, mainly by fibrinolytic systems and matrix metalloproteases (MMPs), may be disturbed in these patients. The causes underlying the deregulation of ECM remodeling that occurs in these patients are not, however, clear. We investigated molecular mechanisms responsible for this dysfunction of ECM remodeling process, comparing it to normal skin. Collagen types, MMP2 and MMP9 expression and activity, interleukins 1β (IL1β) and 6 (IL6), and transcription coactivator PGC-1β expression were analyzed in 16 patients. Ex-obese patients presented increased expression of collagen types III and IV mRNA, increased expression of MMP2, decreased expression and activity of MMP9, and increased expression of PGC-1β in the skin. Inflammation markers IL1β and IL6 mRNA were not different. We have demonstrated that obese patients with extensive weight loss after bariatric surgery have increased expression of PGC-1β in the skin, which can result in a decreased expression and activity of MMP9 and increased collagen types III and IV deposition. These molecular changes may contribute for the formation of saggy skinfolds observed in these patients and impair wound healing.

Purpose We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids. Materials and Methods Multicellular spheroids of mouse MIN6 pancreatic β cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot. Results In all cases, uniformly sized (about 300 μm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic β cells, also increased in their spheroids. Conclusions The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.