Reversion inducing cysteine rich protein with Kazal motifs and cardiovascular diseases: The RECKlessness of adverse remodeling.

The Reversion Inducing Cysteine Rich Protein With Kazal Motifs (RECK) is a glycosylphosphatidylinositol (GPI) anchored membrane-bound regulator of matrix metalloproteinases (MMPs). It is expressed throughout the body and plays a role in extracellular matrix (ECM) homeostasis and inflammation. In initial studies, RECK expression was found to be downregulated in various invasive cancers and associated with poor prognostic outcome. Restoring RECK, however, has been shown to reverse the metastatic phenotype. Downregulation of RECK expression is also reported in non-malignant diseases, such as periodontal disease, renal fibrosis, and myocardial fibrosis. As such, RECK induction has therapeutic potential in several chronic diseases. Mechanistically, RECK negatively regulates various matrixins involved in cell migration, proliferation, and adverse remodeling by targeting the expression and/or activation of multiple MMPs, A Disintegrin And Metalloproteinase Domain-Containing Proteins (ADAMs), and A Disintegrin And Metalloproteinase With Thrombospondin Motifs (ADAMTS). Outside of its role in remodeling, RECK has also been reported to exert anti-inflammatory effects. In cardiac diseases, for example, it has been shown to counteract several downstream effectors of Angiotensin II (Ang-II) that play a role in adverse cardiac and vascular remodeling, such as Interleukin-6 (IL-6)/IL-6 receptor (IL-6R)/glycoprotein 130 (IL-6 signal transducer) signaling and Epidermal Growth Factor Receptor (EGFR) transactivation. This review article focuses on the current understanding of the multifunctional effects of RECK and how its downregulation may contribute to adverse cardiovascular remodeling.

Aldosterone impairs coronary adenosine-mediated vasodilation via reduced functional expression of Ca2+-activated K+ channels.

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

Canagliflozin Inhibits Human Endothelial Cell Proliferation and Tube Formation.

Recent clinical trials revealed that sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly reduce cardiovascular events in type 2 diabetic patients, however, canagliflozin increased limb amputations, an effect not seen with other SGLT2 inhibitors. Since endothelial cell (EC) dysfunction promotes diabetes-associated vascular disease and limb ischemia, we hypothesized that canagliflozin, but not other SGLT2 inhibitors, impairs EC proliferation, migration, and angiogenesis. Treatment of human umbilical vein ECs (HUVECs) with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation. In particular, 10 µM canagliflozin reduced EC proliferation by approximately 45%. The inhibition of EC growth by canagliflozin occurred in the absence of cell death and was associated with diminished DNA synthesis, cell cycle arrest, and a striking decrease in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer partially rescued the proliferative response of HUVECs treated with canagliflozin. A high concentration of canagliflozin (50 µM) modestly inhibited HUVEC migration by 20%, but markedly attenuated their tube formation by 65% and EC sprouting from mouse aortas by 80%. A moderate 20% reduction in HUVEC migration was also observed with a high concentration of empagliflozin (50 µM), while neither empagliflozin nor dapagliflozin affected tube formation by HUVECs. The present study identified canagliflozin as a robust inhibitor of human EC proliferation and tube formation. The anti-proliferative action of canagliflozin occurs in the absence of cell death and is due, in part, to the blockade of cyclin A expression. Notably, these actions are not seen with empagliflozin or dapagliflozin. The ability of canagliflozin to exert these pleiotropic effects on ECs may contribute to the clinical actions of this drug.

Modulation of UVB-induced Carcinogenesis by Activation of Alternative DNA Repair Pathways.

The molecular basis for ultraviolet (UV) light-induced nonmelanoma and melanoma skin cancers centers on cumulative genomic instability caused by inefficient DNA repair of dipyrimidine photoproducts. Inefficient DNA repair and subsequent translesion replication past these DNA lesions generate distinct molecular signatures of tandem CC to TT and C to T transitions at dipyrimidine sites. Since previous efforts to develop experimental strategies to enhance the repair capacity of basal keratinocytes have been limited, we have engineered the N-terminally truncated form (Δ228) UV endonuclease (UVDE) from Schizosaccharomyces pombe to include a TAT cell-penetrating peptide sequence with or without a nuclear localization signal (NLS): UVDE-TAT and UVDE-NLS-TAT. Further, a NLS was engineered onto a pyrimidine dimer glycosylase from Paramecium bursaria chlorella virus-1 (cv-pdg-NLS). Purified enzymes were encapsulated into liposomes and topically delivered to the dorsal surface of SKH1 hairless mice in a UVB-induced carcinogenesis study. Total tumor burden was significantly reduced in mice receiving either UVDE-TAT or UVDE-NLS-TAT versus control empty liposomes and time to death was significantly reduced with the UVDE-NLS-TAT. These data suggest that efficient delivery of exogenous enzymes for the initiation of repair of UVB-induced DNA damage may protect from UVB induction of squamous and basal cell carcinomas.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Feline B7.1 and B7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1-15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.

A model of venous return while utilizing vacuum assist during cardiopulmonary bypass.

In order for vacuum-assisted venous return (VAVR) to be used safely and efficiently during cardiopulmonary bypass (CPB), a full understanding of venous return is necessary. The focus of this work was to use the concepts of energy conservation and viscous energy dissipation in the development of a theoretical model of venous return utilizing vacuum assist. The effectiveness and accuracy of this model has been verified through in vitro laboratory investigations and statistical analysis. Although VAVR can provide higher flows through smaller venous cannula, vacuum assist may lead to increased levels of wall shear stress as shown in this work. The clinical implications of VAVR have yet to be investigated, but may lead to an exacerbation of the detrimental effects of CPB during cardiac surgery.