BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFß to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFß, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFß-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFß1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFß1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFß showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFß, as well as TGFß receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFß or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFß released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

Stem cell mechanisms during left ventricular remodeling post-myocardial infarction: Repair and regeneration.

Post-myocardial infarction (MI), the left ventricle (LV) undergoes a series of events collectively referred to as remodeling. As a result, damaged myocardium is replaced with fibrotic tissue consequently leading to contractile dysfunction and ultimately heart failure. LV remodeling post-MI includes inflammatory, fibrotic, and neovascularization responses that involve regulated cell recruitment and function. Stem cells (SCs) have been transplanted post-MI for treatment of LV remodeling and shown to improve LV function by reduction in scar tissue formation in humans and animal models of MI. The promising results obtained from the application of SCs post-MI have sparked a massive effort to identify the optimal SC for regeneration of cardiomyocytes and the paradigm for clinical applications. Although SC transplantations are generally associated with new tissue formation, SCs also secrete cytokines, chemokines and growth factors that robustly regulate cell behavior in a paracrine fashion during the remodeling process. In this review, the different types of SCs used for cardiomyogenesis, markers of differentiation, paracrine factor secretion, and strategies for cell recruitment and delivery are addressed.

Transcriptional regulation of proteoglycan 4 by 17ß-estradiol in immortalized baboon temporomandibular joint disc cells.

Temporomandibular joint disorders (TMDs) affect a significant portion of the population of the USA, with the majority of those seeking treatment being women of childbearing age. Owing to this striking sexual dimorphism it has been postulated that sex hormones play a role in the maintenance of normal temporomandibular joint (TMJ) function. Proteoglycan 4 (PRG4) is a secreted lubricating molecule required for maintaining low frictional levels within articular joints; however, its role in the TMJ is not well characterized. In this study we describe the development of immortalized baboon cells isolated from specific regions of the TMJ disc and their use in the investigation of PRG4 expression and localization patterns in the TMJ. We identified conserved estrogen response elements within the 5' flanking region of the PRG4 gene of several species, and found that treatment of baboon TMJ disc cells with estrogen led to reduced PRG4 promoter activity and reduced expression of PRG4 mRNA in vitro. The observed negative regulation of PRG4 by estrogen could lead to increased friction and degradation of joint components over time. This study, for the first time, provides evidence of the regulatory potential of estrogen on PRG4 gene expression and suggests a novel etiology for the gender disparity observed among TMD patients.

Differences in the magnitude of long-term potentiation produced by theta burst and high frequency stimulation protocols matched in stimulus number.

Theta-burst stimulation (TBS: four pulses at 100 Hz repeated with 200 ms inter-burst-intervals) and another commonly used high-frequency stimulation protocol (HFS: 1 s burst of equally spaced pulses at 100 Hz) were compared for the magnitude of LTP produced in rat hippocampal slices. The total number of pulses applied during tetanus (TET) was either 40, 100, 200, or 300. In a conventional analysis of the last 10 min of the post-TET period, a two-way ANOVA revealed no difference either in LTP of the field excitatory post-synaptic potential (fEPSP) between TBS and HFS or differences across pulse number at 40, 100, or 200 pulses. At 300 pulses, there was a significant main effect by pulse number but not by protocol. A linear regression analysis showed that stimulation protocol accounted for only about 10% of the change in magnitude while pulse number contributed to 30% of the change. However, when an extended analysis of the same data was performed across the entire post-TET period with a repeated-measure ANOVA, a small but persistent increase in TBS over HFS at 200 pulses was significant. A difference between TBS and HFS at 300 pulses that occurred only during the early phase of LTP was also significant. These results suggest that, over a range of stimuli, the number of pulses in an induction protocol, rather than the pattern of stimulation, determines the magnitude of late phase LTP, while TBS produces greater potentiation than HFS in the early phase of LTP with higher TET number.

A focal adhesion-like process underlies induction of long-term potentiation in the Schaffer collateral-CA1 region of the hippocampus.

In the series of experiments reported here we provide evidence that a focal adhesion-like process underlies the induction of long-term potentiation (LTP) in the Schaffer Collateral-CA1 projection in the hippocampus. Here we show that an integrin binding peptide (RGD) impairs induction of Schaffer Collateral-CA1 LTP in hippocampal slice preparations in vitro. The heparin-binding peptide that binds heparan sulfate proteoglycan (HSPG) and blocks the formation of focal adhesions also impairs induction of LTP. Either the integrin-binding peptide or heparin-binding peptide reduces LTP partially. However, when the two peptides were administered simultaneously, there was no LTP 1 hour after induction. This indicates that these two molecules might function together and that a focal adhesion-like process might be involved in the induction of LTP. Additionally,we report that the RGD effect on LTP is time dependent and occurs only in the first few minutes following LTP induction, that the binding of the RGD peptide in CA1 stratum radiatum increases after LTP induction and that this increased binding depends on Ca(2+). Using electron microscopy we show that integrins are present in synapses.