Caroline J. Taylor

Induction of Heme Oxygenase-1 In Vivo Suppresses NADPH Oxidase–Derived Oxidative Stress

Our previous studies suggest that heme oxygenase (HO)-1 induction and/or subsequent bilirubin generation in endothelial cells may suppress superoxide generation of from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. In this study, we examined the consequence of HO-1 induction in vivo on NADPH oxidase activity. Three doses of hemin (25 mg · kg−1, IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg · kg−1, IP), were given to apolipoprotein E–deficient mice, which display vascular oxidative stress. Hemin treatment increased HO-1 expression and activity in aorta (undetectable at baseline) and kidney (by 3-fold) and significantly reduced both NADPH oxidase activity (by ≈25% to 50%) and superoxide generation in situ. The increase in HO-1 activity and inhibition of NADPH oxidase activity by hemin were reversed by tin protoporphyrin-IX and were not associated with changes in Nox2 or Nox4 protein levels. Hemin also reduced plasma F2-isoprostane levels by 23%. The inhibition of NADPH oxidase activity by hemin in the aorta was mimicked by bilirubin in vitro (0.01 to 1 &mgr;mol/L). Bilirubin also concentration-dependently reduced NADPH oxidase–dependent superoxide production stimulated by angiotensin II in rat vascular smooth muscle cells and by phorbol 12-myristate 13-acetate in human neutrophil-like HL-60 cells. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH-stimulated superoxide production. Thus, systemic expression of HO-1 suppresses NADPH oxidase activity by mechanisms at least partly mediated by the bile pigment bilirubin, thereby reducing oxidative stress.

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

Fluid secretion across the blood–brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO3−, which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pHi). To analyse the transporters that may be involved in the maintenance of pHi and hence secretion of HCO3−, we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO2/HCO3−. pHi in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO3− than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO3− were similar. In the nominal absence of HCO3− the rate of acid loading was increased equally by removal of external Na+ or by inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO3− the increase in the rate of acid loading when Na+ was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na+ and HCO3−. Removal of Cl− in the presence of HCO3− produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na+ following removal of Cl− increased the rate of acid gain. In the presence of HCO3− and initial presence of Na+ and Cl−, DIDS inhibited the changes in pHi produced by removal of either Na+ or Cl−. These are the expected results if these cells possess an AE‐like Cl−/HCO3− exchanger, a ‘channel‐like’ permeability allowing slow influx of acid (or efflux of HCO3−), a NBC‐like Cl−‐independent Na+−HCO3− cotransporter, and a NHE‐like Na+/H+ exchanger. The in vitro rates of HCO3− loading via the Na+−HCO3− cotransporter could, if the transporter is located on the apical, blood‐facing side of the cells, account for the net secretion of HCO3− into the brain.

Hypoxia‐inducible factor 1α modulates adhesion, migration, and FAK phosphorylation in vascular smooth muscle cells

Hypoxia promotes angiogenesis by modulating the transcriptional regulator hypoxia‐inducible factor 1α (HIF‐1α). HIF‐1α is a master regulator of the hypoxic response, and its proangiogenic activities include, but are not limited to, regulation of vascular endothelial growth factor (VEGF). The remodeling of the vasculature during angiogenesis requires an initial destabilization step, which facilitates endothelial sprouting, followed by vessel growth, and restabilization through investment of smooth muscle cells. The complex dynamics of hypoxia‐induced angiogenesis prompted us to investigate what aspects of this multi‐step process are regulated by HIF‐1α. To do so, we analyzed the molecular properties of aortic and coronary artery smooth muscle cells in response to forced expression of HIF‐1α, and by treatment with cobalt chloride, which mimics hypoxia. Our results demonstrate that HIF‐1α causes a marked reduction in the ability of smooth muscle cells to migrate and adhere to extracellular matrices. Analysis of focal adhesion proteins showed no significant difference in expression or localization of vinculin or focal adhesion kinase (FAK). However, investigation of FAK phosphorylation, a critical mediator of adhesion and migration, revealed tyrosine phosphorylation of FAK is diminished in the presence of HIF‐1α and cobalt chloride. These results indicate that during hypoxia‐induced vessel remodeling, HIF‐1α functions to dampen adhesion and migration of smooth muscle cells by modulating FAK activity. We suggest that HIF‐1α expression in smooth muscle cells may augment vessel sprouting by loosening smooth muscle cell attachments to the basement membrane and endothelial cells. J. Cell. Biochem.

An Adipoinductive Role of Inflammation in Adipose Tissue Engineering: Key Factors in the Early Development of Engineered Soft Tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

Modulation of LPA Receptor Expression in the Human Brain Following Neurotrauma

Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR2 and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR1 colocalized with astrocytes and that LPAR2 is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.

Prostacyclin receptor suppresses cardiac fibrosis: Role of CREB phosphorylation

Cardiac fibrosis is a consequence of many cardiovascular diseases and contributes to impaired ventricular function. Activation of the prostacyclin receptor (IP) protects against cardiac fibrosis, but the molecular mechanisms are not totally understood. Using mouse cardiac fibroblasts, we found that IP activation with cicaprost suppressed expression of collagen I and other target genes of transforming growth factor-beta. This effect of cicaprost was unlikely to be mediated by inhibition of the Smad2/3 or mitogen-activated protein kinase (MAPK) activities, but was associated with cAMP elevation and phosphorylation of the transcription factor cAMP response element binding protein (CREB). Expression of a non-phosphorylated CREB mutant suppressed the inhibitory effect of cicaprost. It appears that phosphorylated CREB binds to and sequestrates the transcription coactivator CBP/p300 from binding to Smad. Inhibition of the intrinsic histone acetyl-transferase activity of CBP/p300 with garcinol significantly suppressed collagen I expression in fibroblasts. Using apolipoprotein E and IP double knockout mouse, we demonstrated that endogenous prostacyclin/IP signaling had an inhibitory effect on angiotensin II-induced cardiac fibrosis under hypercholesterolemic conditions. Taken together, our results suggest that the prostacyclin/IP pathway suppresses cardiac fibrosis, at least partly, by inducing CREB phosphorylation.

Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis.

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms

Annexin Peptide Ac2-26 Suppresses TNFα-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells

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