Curvature facilitates podocyte culture in a biomimetic platform.

Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels.

Diabetic wound regeneration using peptide-modified hydrogels to target re-epithelialization.

There is a clinical need for new, more effective treatments for chronic wounds in diabetic patients. Lack of epithelial cell migration is a hallmark of nonhealing wounds, and diabetes often involves endothelial dysfunction. Therefore, targeting re-epithelialization, which mainly involves keratinocytes, may improve therapeutic outcomes of current treatments.

PI3K Phosphorylation Is Linked to Improved Electrical Excitability in an In Vitro Engineered Heart Tissue Disease Model System.

Myocardial infarction, a prevalent cardiovascular disease, is associated with cardiomyocyte cell death, and eventually heart failure. Cardiac tissue engineering has provided hopes for alternative treatment options, and high-fidelity tissue models for drug discovery. The signal transduction mechanisms relayed in response to mechanoelectrical (physical) stimulation or biochemical stimulation (hormones, cytokines, or drugs) in engineered heart tissues (EHTs) are poorly understood.

Hydrogels with integrin-binding angiopoietin-1-derived peptide, QHREDGS, for treatment of acute myocardial infarction.

Background: Hydrogels are being actively investigated for direct delivery of cells or bioactive molecules to the heart after myocardial infarction (MI) to prevent cardiac functional loss. We postulate that immobilization of the prosurvival angiopoietin-1-derived peptide, QHREDGS, to a chitosan-collagen hydrogel could produce a clinically translatable thermoresponsive hydrogel to attenuate post-MI cardiac remodeling.

Methods And Results: In a rat MI model, QHREDGS-conjugated hydrogel (QHG213H), control gel, or PBS was injected into the peri-infarct/MI zone.

Inhibition of apoptosis in human induced pluripotent stem cells during expansion in a defined culture using angiopoietin-1 derived peptide QHREDGS.

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival, self-renewal, and differentiation. Thus, hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture.

Bioreactor for modulation of cardiac microtissue phenotype by combined static stretch and electrical stimulation.

We describe here a bioreactor capable of applying electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.

Intercellular interactions between mast cells and fibroblasts promote pro-inflammatory signaling.

The mechanisms that mediate acute exacerbations in chronic inflammatory diseases are not understood. As IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions, we investigated the role of fibroblast-mast cell interactions in short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC).

Impact of matrix metalloproteinases on inhibition of mineralization by fetuin.

Background And Objective: Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro.

Importance of protein-tyrosine phosphatase-alpha catalytic domains for interactions with SHP-2 and interleukin-1-induced matrix metalloproteinase-3 expression.

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs). We examined adhesion-restricted signaling pathways that enable IL-1-induced MMP release in human gingival and murine fibroblasts. Of the seven MMPs and three tissue inhibitors of MMPs screened, IL-1 enhanced release only of MMP3 when cells formed focal adhesions.

Methylglyoxal-modified collagen promotes myofibroblast differentiation.

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO).

Novel function of PERK as a mediator of force-induced apoptosis.

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads.

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves.

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.

Phosphatidylinositol-4,5 bisphosphate produced by PIP5KIgamma regulates gelsolin, actin assembly, and adhesion strength of N-cadherin junctions.

Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin-mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCdelta showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1.

Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway.

In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle alpha-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho-Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.

Methylglyoxal inhibits the binding step of collagen phagocytosis.

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues.

Phosphorylation of N-cadherin-associated cortactin by Fer kinase regulates N-cadherin mobility and intercellular adhesion strength.

Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. We show that the nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and that this process is involved in mediating intercellular adhesion strength.

Collagen phagocytosis by fibroblasts is regulated by decorin.

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts.

Mechanical force activates eIF-2alpha phospho-kinases in fibroblast.

Mechanical forces can induce differentiation of fibroblasts into myofibroblasts, a process which requires activation of the MAP kinase p38. Currently, the identification of other phospho-kinases involved in myofibroblast differentiation has not been explored. We applied static tensile forces to rat cardiac fibroblasts via collagen-coated magnetite beads and examined activation of protein phospho-kinases by the Kinexus phospho-antibody screening system.

Smooth muscle actin determines mechanical force-induced p38 activation.

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.

Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts.

The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 microM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions.

Regulation of intercellular adhesion strength in fibroblasts.

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes.

Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases.

Background: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete.

Activation of neutrophil collagenase in periodontitis.

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate.

Modulation of plasminogen activators and plasminogen activator inhibitors by TGF-beta, IL-1 alpha and EGF in fetal rat calvaria cells at different times of culture.

Fetal rat calvaria cells (RC cells) grown in long term culture in the presence of ascorbic acid and organic phosphate proliferate and differentiate to form mineralized nodules of bone. Since transforming growth factor beta (TGF-beta), interleukin 1-alpha (IL-1 alpha) and epidermal growth factor (EGF) affect both bone resorption and bone formation, we have studied the ability of these growth factors to affect plasminogen activators and plasminogen activator inhibitors release by RC cells at different times throughout this proliferation/differentiation sequence. Cultures in log phase growth (day 4), when first multilayering (day 7) and when bone nodules were forming (day 13) were exposed to either TGF-beta, IL-1 alpha, EGF or vehicle.

Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha.

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa.