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Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK) and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell permeant phosphopeptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and also in multiple clinically relevant injury models. Anisomycin (Aniso) increased p38 MAPK phosphorylation and reduced endothelial‐dependent relaxation in RA. Treatment with NiPp prevented Ansio‐induced reduction in endothelial function and increases in p38 MAPK phosphorylation. NiPp treatment also restored endothelial function after stretch injury (subfailure stretch), treatment with acidic Normal Saline (NS), and P2X7R activation with 2′(3′)‐O‐(4‐Benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP). Aged, diseased, human saphenous vein (HSV) remnants obtained from patients undergoing coronary bypass surgical procedures have impaired endothelial function. Treatment of these HSV segments with NiPp improved endothelial‐dependent relaxation. Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. The NiPp peptide may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.

Severe acute asthma, defined as asthma that is refractory to current anti-inflammatory agents and bronchodilators, accounts for an estimated two-million emergency department visits annually. β2-adrenergic receptor (β2AR) agonists (β-agonists) are widely used for the treatment of bronchospasm associated with acute asthma attacks, however, some patients develop refractory responses to long term usage of β –agonists. It also causes adverse effects in patients with a genetic polymorphism (Arg/Arg genotype, about a sixth of the US population) in the adrenergic receptor creating a need for additional therapeutics for managing asthma. We have shown earlier that β-agonist induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of HSP20. In this study we evaluate the effect of a transducible phosphopeptide mimetic of HSP20 protein (P20 peptide) on the relaxation of human ASM. Freshly isolated human ASM was pretreated with either a β-agonist, isoproterenol (ISO), or the P-2...

The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10−7M), NO (10−10–10−6M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. Zaprinast (10−4M), increased NO-stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10−5M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10−10–10−8M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10−7–10−6M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10−7–10−5M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.