Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.

A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCo3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.

A disaccharide that inhibits tumor necrosis factor alpha is formed from the extracellular matrix by the enzyme heparanase.

The activation of T cells by antigens or mitogens leads to the secretion of cytokines and enzymes that shape the inflammatory response. Among these molecular mediators of inflammation is a heparanase enzyme that degrades the heparan sulfate scaffold of the extracellular matrix (ECM). Activated T cells use heparanase to penetrate the ECM and gain access to the tissues. We now report that among the breakdown products of the ECM generated by heparanase is a trisulfated disaccharide that can inhibit delayed-type hypersensitivity (DTH) in mice. This inhibition of T-cell mediated inflammation in vivo was associated with an inhibitory effect of the disaccharide on the production of biologically active tumor necrosis factor alpha (TNF-alpha) by activated T cells in vitro; the trisulfated disaccharide did not affect T-cell viability or responsiveness generally. Both the in vivo and in vitro effects of the disaccharide manifested a bell-shaped dose-response curve. The inhibitory effects of the trisulfated disaccharide were lost if the sulfate groups were removed. Thus, the disaccharide, which may be a natural product of inflammation, can regulate the functional nature of the response by the T cell to activation. Such a feedback control mechanism could enable the T cell to assess the extent of tissue degradation and adjust its behavior accordingly.

Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix

During puberty, pregnancy, lactation and postlactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.

Genes for the majority of group A streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease

Background. The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. Methods. In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. Results. Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. Conclusions. Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/ or host factors.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta 2 and gamma 1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha 1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha 1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha 1, laminins beta 2 and gamma 1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Vesicoureteral reflux and the extracellular matrix connection.

Primary vesicoureteral reflux (VUR) is a common pediatric condition due to a developmental defect in the ureterovesical junction. The prevalence of VUR among individuals with connective tissue disorders, as well as the importance of the ureter and bladder wall musculature for the anti-reflux mechanism, suggest that defects in the extracellular matrix (ECM) within the ureterovesical junction may result in VUR. This review will discuss the function of the smooth muscle and its supporting ECM microenvironment with respect to VUR, and explore the association of VUR with mutations in ECM-related genes.

The extracellular matrix contributes to mechanotransduction in uterine fibroids.

The role of the extracellular matrix (ECM) and mechanotransduction as an important signaling factor in the human uterus is just beginning to be appreciated. The ECM is not only the substance that surrounds cells, but ECM stiffness will either compress cells or stretch them resulting in signals converted into chemical changes within the cell, depending on the amount of collagen, cross-linking, and hydration, as well as other ECM components. In this review we present evidence that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (1) resistance to apoptosis leading to the persistence of cells and (2) secretion of collagen and other components of the ECM such a proteoglycans by those cells leading to abundant disposition of highly cross-linked, disoriented, and often widely dispersed collagen fibrils. Fibrosis affects cell growth by mechanotransduction, the dynamic signaling system whereby mechanical forces initiate chemical signaling in cells. Data indicate that the structurally disordered and abnormally formed ECM of uterine fibroids contributes to fibroid formation and growth. An appreciation of the critical role of ECM stiffness to fibroid growth may lead to new strategies for treatment of this common disease.

Modest elevation in BNP in asymptomatic hypertensive patients reflects sub-clinical cardiac remodeling, inflammation and extracellular matrix changes

In asymptomatic subjects B-type natriuretic peptide (BNP) is associated with adverse cardiovascular outcomes even at levels well below contemporary thresholds used for the diagnosis of heart failure. The mechanisms behind these observations are unclear. We examined the hypothesis that in an asymptomatic hypertensive population BNP would be associated with sub-clinical evidence of cardiac remodeling, inflammation and extracellular matrix (ECM) alterations. We performed transthoracic echocardiography and sampled coronary sinus (CS) and peripheral serum from patients with low (n = 14) and high BNP (n = 27). Peripheral BNP was closely associated with CS levels (r = 0.92, p<0.001). CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP (r = 0.44, p = 0.008), CITP (r = 0.35, p = 0.03) and PIIINP (r = 0.35, p = 0.001), and with CS levels of inflammatory cytokines including: TNF-α (r = 0.49, p = 0.002), IL-6 (r = 0.35, p = 0.04), and IL-8 (r = 0.54, p<0.001). The high BNP group had greater CS expression of fibro-inflammatory biomarkers including: CITP (3.8±0.7 versus 5.1±1.9, p = 0.007), TNF-α (3.2±0.5 versus 3.7±1.1, p = 003), IL-6 (1.9±1.3 versus 3.4±2.7, p = 0.02) and hsCRP (1.2±1.1 versus 2.4±1.1, p = 0.04), and greater left ventricular mass index (97±20 versus 118±26 g/m(2), p = 0.03) and left atrial volume index (18±2 versus 21±4, p = 0.008). Our data provide insight into the mechanisms behind the observed negative prognostic impact of modest elevations in BNP and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and ECM alterations.

The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.

A cell migration device that maintains a defined surface with no cellular damage during wound edge generation

Studying the rate of cell migration provides insight into fundamental cell biology as well as a tool to assess the functionality of synthetic surfaces and soluble environments used in tissue engineering. The traditional tools used to study cell migration include the fence and wound healing assays. In this paper we describe the development of a microchannel based device for the study of cell migration on defined surfaces. We demonstrate that this device provides a superior tool, relative to the previously mentioned assays, for assessing the propagation rate of cell wave fronts. The significant advantage provided by this technology is the ability to maintain a virgin surface prior to the commencement of the cell migration assay. Here, the device is used to assess rates of mouse fibroblasts (NIH 3T3) and human osteosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. The device design enables rapid and simplistic assessment of multiple repeats on a single chip, where surfaces have not been previously exposed to cells or cellular secretions.