Stimulation Of Extracellular Matrix Synthesis Research Articles

Human beta defensin levels and vaginal microbiome composition in post-menopausal women diagnosed with lichen sclerosus

Human beta defensins (hBDs) may play an important role in the progression of lichen sclerosus (LS), due to their ability to induce excessive stimulation of extracellular matrix synthesis and fibroblast activation. The genetic ability of the individual to produce defensins, the presence of microbes influencing defensin production, and the sensitivity of microbes to defensins together regulate the formation of an ever-changing balance between defensin levels and microbiome composition. We investigated the potential differences in postmenopausal vaginal microbiome composition and vaginal hBD levels in LS patients compared to non-LS controls. LS patients exhibited significantly lower levels of hBD1 (p = 0.0003), and significantly higher levels of hBD2 (p = 0.0359) and hBD3 (p = 0.0002), compared to the control group. The microbiome of the LS patients was dominated by possibly harmful bacteria including Lactobacillus iners, Streptococcus anginosus or Gardnerella vaginalis known to initiate direct or indirect damage by increasing defensin level production. Our observations highlight that correcting the composition of the microbiome may be applicable in supplementary LS therapy by targeting the restoration of the beneficial flora that does not increase hBD2-3 production.

Induction of bone marrow mesenchymal stem cell chondrogenesis following short-term suspension culture

The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGFβ). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four- to ninefold increase in extracellular matrix (ECM) synthesis in TGFβ-free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan-rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGFβ exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion-conditioned MSCs without additional TGFβ exposure was less than samples maintained in TGFβ throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early-stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short-term culture.

Targeting pleiotropin to treat osteoarthritis

Pleiotropin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. Although PTN is abundant in fetal or juvenile cartilage, it is undectable in mature cartilage. However, PTN is re-expressed in chondrocytes in early stages of osteoarthritis where it is detectable in situ and in synovial fluids from patients. PTN enhances chondrogenesis by stimulation of extra-cellular matrix synthesis, reduction of degrading matrix metalloproteases and induction of their inhibitors; PTN also slightly reduces pro-inflammatory factors, such as nitric oxide and vascular endothelial growth factor. Furthermore, PTN stimulates chondrocyte clustering and proliferation. Thus, PTN appears to mediate repair and protective processes in osteoarthritic cartilage and appears to be a promising factor to treat osteoarthritis.

Direct renin inhibitors: the dawn of a new era, or just a variation on a theme?

Numerous studies have demonstrated that the renin-angiotensin-aldosterone system (RAAS) plays animportant role in the progression of chronic kidneydisease (CKD). Angiotensin (Ang) II generates intra-renal haemodynamic and inflammatory changes thatpromote proteinuria, growth of glomerular andtubular cells, inhibition of NO synthesis, stimulationof extracellular matrix synthesis and induction ofchemokines, reactive oxygen species and apoptosis[1]. In addition, in animal models of renal diseases,aldosterone is also involved in endothelial dysfunction,inflammation, proteinuria and fibrosis [2]. In clinicaltrials, treatment with angiotensin-converting enzyme(ACE) inhibitors and Ang II AT1 receptor blockers(ARBs) was proved to slow down the evolution of bothdiabetic and non-diabetic nephropathies and, there-fore, it is currently regarded as the cornerstone of whatwe call ‘nephroprotection’.A controversial issue is whether these agents aresuperior to other antihypertensive drugs in terms ofnephroprotection—in other words, do they possessso-called ‘pleiotropic’ effects? A large meta-analysis [3]suggested that the benefit seen with ACE inhibitorswas related to the blood pressure (BP) decrease alone.The truth, as Sarafidis et al. [4] pointed out in a veryrecent review, is that, indeed, ACE inhibitors exhibit aBP-independent renoprotective effect, but only inpatients with proteinuria and advanced CKD(i.e. stage 3 or 4), and also that the higher the baselineurine protein excretion and its subsequent decrease, themore pronounced the beneficial effect of these drugs islikely to be [4].However, it is not sufficiently appreciated that, dueto the remarkable complexity of the RAAS, neitherACE inhibitors nor ARBs alone can completelysuppress its activity (Figure 1). The main limitationsof these agents in that regard can be summarized asfollows:(i) A substantial proportion of Ang II is generated inthe kidneys from Ang I via non-ACE pathways,such as chymase [5], which are not susceptible tothe actions of ACE inhibitors.(ii) ACE inhibition and ARB, by interrupting nega-tive feedback-loops, result in high plasma (andmost likely tissue-) renin concentrations and highplasma renin activity (PRA), which may over-come the drugs’ effectiveness. The reactivation ofAng II (or ‘Ang II escape’) has been describedwith both ACE inhibitors and ARBs [6]; how-ever, its clinical significance is not yet fullyunderstood.(iii) Renin can also bind to specific receptors in themesangium and in the subendothelium of therenal arteries. This binding leads on one hand, toa substantial increase in the catalytic efficiency ofconversion of angiotensinogen to Ang I, and, onthe other hand, to stimulation of mitogen-activated protein kinases, ERK-1 and ERK-2[7]. In animal models of nephrotic syndrome,treatment with ACE inhibitors was associatedwith a reduction in glomerular injury andproteinuria, but also with tubulo-interstitiallesions [8]. These findings support a directprofibrotic role for renin, independent of itsAng-generating effect.(iv) ACE inhibitors induce an acute decrease inplasma aldosterone. This effect, however, isoften only transient and followed by a progres-sive rise in aldosterone levels that ultimately, withlong-term ACE inhibition, can reach normal oreven elevated concentrations. This ‘aldosterone

Involvement of the constitutive complex formation of c‐Ski/SnoN with smads in the impaired negative feedback regulation of transforming growth factor β signaling in scleroderma fibroblasts

The principal effect of transforming growth factor beta1 (TGFbeta1) on mesenchymal cells is its stimulation of extracellular matrix synthesis. Previous reports indicated the significance of the autocrine TGFbeta loop in the pathogenesis of scleroderma. The aim of this study was to examine c-Ski and SnoN, principal molecules in the negative regulation of TGFbeta signaling, to further understand the autocrine TGFbeta loop in scleroderma. Levels of expression of c-Ski/SnoN on cultured normal and scleroderma fibroblasts were determined by Western blotting, Northern blotting, and immunohistochemical staining. To determine the protein-protein interaction between c-Ski/SnoN, Smads, and p300, immunoprecipitation was performed. A transient transfection assay was performed to measure promoter activity of the alpha2(I) collagen gene and the 3TP-Lux plasmid construct. Scleroderma fibroblasts exhibited increased c-Ski/SnoN levels compared with normal fibroblasts, both in vivo and in vitro. Although c-Ski/SnoN constitutively formed a complex with Smads by immunoprecipitation, the inhibitory effect of c-Ski/SnoN on the promoter activity of human alpha2(I) collagen and 3TP-Lux was impaired in scleroderma fibroblasts. Immunoprecipitation analyses also revealed that overexpressed c-Ski/SnoN could not compete with p300 in these cells. These results indicate that impaired competition with p300 is the possible cause of dysfunction of c-Ski/SnoN in scleroderma fibroblasts and that this might contribute to maintenance of the autocrine TGFbeta loop in this disease.

Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury

Recent evidence indicates that transforming growth factor-beta 1 (TGF-beta 1) plays an important role in renal fibrosis via stimulation of extracellular matrix synthesis. The present study was undertaken to investigate the role of angiotensin II type I receptor (AT1 receptor) in hypertension-induced renal injury. Twenty-two-week-old stroke-prone spontaneously hypertensive rats (SHRSP), which had established hypertension and moderate renal damage, were orally given TCV-116, a selective non-peptide AT1 receptor antagonist (0.1, 1 or 10 mg/kg/day), enalapril (10 mg/kg/day) or vehicle once a day for 10 weeks. At the end point of the treatment, we examined renal function, the gene expressions of TGF-beta 1 and extracellular matrix components in the interstitium [collagen types I (COI) and III (COIII), fibronectin (FN)] and the basement membrane (COIV and laminin), and renal microscopic morphology in rats aged 32 weeks. In vehicle-treated 32 week-old SHRSP with renal dysfunction and nephrosclerosis, renal mRNA levels for TGF-beta 1, COI, COIII, FN, COIV were all several-fold higher than in WKY. Thus, renal TGF-beta 1 gene expression was enhanced in SHRSP, which may contribute to the increased renal expressions of COI, COIII, FN, COIV in SHRSP. Treatment with TCV-116 (0.1 mg/kg/day) in SHRSP, in spite of no reduction of blood pressure, decreased renal mRNA levels for TGF-beta 1, COI, COIII, FN, COIV, being accompanied by the significant decrease in urinary protein and albumin excretion, blood urea nitrogen and plasma creatinine. Treatment with TCV-116 (10 mg/kg/day) in SHRSP decreased mRNAs for TGF-beta 1, COI, COIII, FN and COIV to almost the same levels as WKY, being associated with normalization of urinary protein and albumin excretion and the prevention of nephrosclerosis, as judged by microscopic histological observations. On the other hand, the effects of enalapril (10 mg/kg/day) on the above mentioned mRNA levels, renal function and renal morphology were weaker than those of TCV-116 (10 mg/kg/day) and were as much as TCV-116 (1 mg/kg/day). These results suggest that independently of hypotensive action, AT1 receptor antagonist has a potent renal protective effect by inhibiting the gene expression of renal TGF-beta 1 and extracellular matrix components.

Epidermal growth factor and transforming growth factor alpha regulate extracellular matrix production by embryonic mouse palatal mesenchymal cells cultured on a variety of substrata.

Mouse embryonic palatal mesenchymal (MEPM) cells were cultured either on plastic tissue culture dishes or on the surface of three-dimensional collagen gels or within collagen gel matrices in DMEM/F12 medium containing 2.5% donor calf serum. MEPM cells proliferated exponentially when cultured on collagen or on plastic. Cells cultured within collagen gels did not proliferate but remained viable. Addition of 10 ng/ml epidermal growth factor (EGF) or transforming growth factor alpha (TGFα) stimulated the proliferation of those cells cultured on plastic or on collagen but not those cultured within collagen gels. Immunocytochemical analysis revealed that MEPM cells synthesise collagen types I, III, IV, V, VI and IX; fibronectin, heparan sulphate proteoglycan, laminin and tenascin in vitro. These molecules are all present in the developing palate in vivo. EGF and TGFα produced a generalised stimulation of extracellular matrix (ECM) synthesis by MEPM cells in vitro. Biochemical analysis indicated that cells cultured within collagen gels had the highest intrinsic rate of protein synthesis. On all substrata neither EGF nor TGFα markedly altered the types of ECM molecules synthesised but rather caused a general increase in the total amount produced. This stimulation was most marked where the cells were cultured within collagen gels. The lack of stimulation of proliferation of MEPM cells cultured within collagen gels (i.e. in a physiologically-relevant environment) by EGF or TGFα together with the marked stimulation of ECM synthesis suggests that these factors may act as differentiation signals via their effects on ECM production.

Effects of alprazolam and triazolam on the synaptosomal calcium levels and glutamate release

Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury. Recent evidence indicates that transforming growth factor-β1 (TGF-β1) plays an important role in renal fibrosis via stimulation of extracellular matrix synthesis. The present study was undertaken to investigate the role of angiotensin II type I receptor (AT1 receptor) in hypertension-induced renal injury. Twenty-two-week-old stroke-prone spontaneously hypertensive rats (SHRSP), which had established hypertension and moderate renal damage, were orally given TCV-116, a selective non-peptide AT1 receptor antagonist (0.1, 1 or 10 mg/kg/day), enalapril (10 mg/kg/day) or vehicle once a day for 10 weeks. At the end point of the treatment, we examined renal function, the gene expressions of TGF-β1 and extracellular matrix components in the interstitium [collagen types I (COI) and III (COIII), fibronectin (FN)] and the basement membrane (COIV and laminin), and renal microscopic morphology in rats aged 32 weeks. In vehicle-treated 32 week-old SHRSP with renal dysfunction and nephrosclerosis, renal mRNA levels for TGF-β1, COI, COIII, FN, COIV were all several-fold higher than in WKY. Thus, renal TGF-β1 gene expression was enhanced in SHRSP, which may contribute to the increased renal expressions of COI, COIII, FN, COIV in SHRSP. Treatment with TCV-116 (0.1 mg/kg/day) in SHRSP, in spite of no reduction of blood pressure, decreased renal mRNA levels for TGF-β1, COI, COIII, FN, COIV, being accompanied by the significant decrease in urinary protein and albumin excretion, blood urea nitrogen and plasma creatinine. Treatment with TCV-116 (10 mg/kg/day) in SHRSP decreased mRNAs for TGF-β1, COI, COIII, FN and COIV to almost the same levels as WKY, being associated with normalization of urinary protein and albumin excretion and the prevention of nephrosclerosis, as judged by microscopic histological observations. On the other hand, the effects of enalapril (10 mg/kg/day) on the above mentioned mRNA levels, renal function and renal morphology were weaker than those of TCV-116 (10 mg/kg/day) and were as much as TCV-116 (1 mg/kg/day). These results suggest that independently of hypotensive action, AT1 receptor antagonist has a potent renal protective effect by inhibiting the gene expression of renal TGF-β1 and extracellular matrix components.

Stimulation of extracellular matrix synthesis in the developing cornea by glycosaminoglycans.

Previously, it was demonstrated that the embryonic corneal epithelium produces the chondroitin sulfate and heparan-sulfate-like compounds and the collagen of the primary corneal stroma. Synthesis of all of these extracellular materials is greatly enhanced in vitro when isolated epithelium is grown on collagenous substrata instead of Millipore filters. We report here that chondroitin sulfate, heparin, and heparan sulfate added to the culture medium at a concentration of 200 mug/ml enhance the synthesis by the epithelium of chondroitin sulfate and heparan-sulfate-like compounds 2-fold, whether or not collagenous substrata are employed. Collagen synthesis is unaffected by adding glycosaminoglycan to the medium. Chondroitin sulfate proteoglycan (chondromucoprotein) has the same stimulatory effect as chondroitin sulfate, but dermatan sulfate and hyaluronate have no measurable effect on glycosaminoglycan production by epithelial cells. Keratan sulfate however, seems to depress glycosaminoglycan synthesis. Thus, in this system, only sulfated polyanions like those produced by the corneal epithelium have a stimulatory effect on glycosaminoglycan synthesis. The results are discussed in terms of how the tissues of the cornea (epithelium, endothelium, keratocytes) may interact by changing the composition of the stromal extracellular matrix.