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Background: Endothelial dysfunction decreases nitric oxide, which leads to the development of intimal hyperplasia and, eventually, vein graft failure. Failed vein grafts have been found to have calcium deposits, enlarged intima, atherosclerotic plaques, and extracellular matrix deposition. This project examined the effect of calcification on endothelial function by measuring the endothelial relaxation and the expression of nitric oxide synthase (eNOS) in saphenous veins. Methods: Freshly collected HSV (58) samples from CABG patients were equilibrated in a muscle bath and phenylephrine-induced contraction and carbachol-induced relaxation (endothelium-dependent) were measured. Vein sections were stained using Verhoeff-Van Gieson (VVG), Movat, and Von Kossa stains. Endothelial coverage was determined by eNOS, and Lectin staining. Calcification, intimal and medial basal thickness, endothelial coverage, and extracellular matrix deposition data were quantified using light microscopy and digital image analysis. Results: Of the 58 veins analyzed, 17 displayed calcification. The average basal intimal thickness of the samples was 65.44 ± 7.99 μm. Presence of calcification in HSV correlated with higher intimal basal thickness (p=0.0092, n=46, panel A), lower endothelial relaxation (p=0.032, n=43, panel B), loss of eNOS expression (p=0.0397, n=29), loss of endothelial coverage (p=0.0178, n=29), and increased extracellular matrix deposition (p=0.0180, n=22, panel C). Conclusions: Calcified veins displayed lowered endothelial relaxation, higher intimal basal thickness, reduced endothelial coverage, reduced eNOS expression, and increased extracellular matrix deposition. Calcification was associated with factors that contribute to intimal hyperplasia formation and vein graft failure.

BACKGROUND: Human saphenous vein (HSV) remains the most widely utilized conduit for vascular bypass procedures. Outcomes remain limited by vein graft failure and intimal hyperplasia. Endothelial function is considered an important determinant of vein graft failure. We have observed significant variability in endothelial function in freshly isolated HSV samples. We therefore evaluated clinical predictors of endothelial function in HSV. Methods: We obtained freshly isolated HSV samples from the operating room immediately following harvest from patients undergoing coronary artery bypass procedures from Vanderbilt University Hospital and the Nashville VA Hospital. We obtained HSV samples prior to any intraoperative manipulations. HSV viability, smooth muscle function, and endothelial-dependent relaxation (EDR) were measured in a muscle bath. We collected the following clinical and demographic information: age, height, weight, gender, ethnicity, medical comorbidities, laboratory values (creatinine, HbA1c, lipids), ejection fraction, preoperative medication regimen, and method of vein graft harvest. We performed a univariate and multivariate analysis of predictors of endothelial function in HSV. Results: HSV samples were obtained from 149 patients. HSV EDR varied from -11% to 63%. Open harvest was employed for 39 HSV samples, compared with endoscopic harvest in 110 HSV samples. On univariate analysis, only open harvest was a statistically significant predictor of endothelial function (p=0.02): mean HSV EDR was 21.5% in HSV samples harvested open, compared with 15.4% in HSV harvested with an endoscopic technique. HSV EDR was also improved with preoperative aspirin use: mean HSV EDR was 18% in patients with preoperative aspirin use compared with 11.6% in patients who were not administered preoperative aspirin in a multivariate model when controlling for method of vein graft harvest (p=0.05). Conclusions: Endoscopic vein graft harvest is associated with endothelial dysfunction in HSV, while preoperative aspirin use is associated with improved endothelial function. Further work will be necessary to determine whether these factors are associated with development of intimal hyperplasia and vein graft failure.

Subarachnoid hemorrhages affect 30,000 people each year and accounts for between 1-7% of all strokes. Nearly 85% of the time that the hemorrhage occurs, it is due to a cerebral aneurysm that leads to delayed vasospasm and stroke. Traditional vasodilators cannot be used because they will cause systemic hypotension leaving an unmet clinical need. HSPB1 is a small heat shock related protein that modulates vasorelaxation, is downregulated in vasospastic vessels, and represents a potential therapeutic modality for the treatment of SAH-induced vasospasm. siRNA against HSPB1 that was encapsulated with a diblock copolymer to enhance uptake, or Rat aortic smooth muscle was tested with a HSPB1 phospho-peptide (HSPB1-p) +/- a polyplex delivery system (polyplexHSPB1). Intracellular calcium was measured concurrently with force using a Fluoroplex sysem. Knockdown of HSPB1 inhibited sodium nitroprusside (SNP)-induced relaxation of rat aorta. In conclusion, HSP20 modulates calcium independent relaxation of vascular smooth muscle and HSPB1-p may represent a novel therapeutic for vasospasm. Tissue treated with HSPB1-p or polyplex HSPB1-p had a dose dependent inhibition of contraction. The polyplex inhibited contraction at a lower dose of HSPB1-p than peptide alone with complete inhibition of contraction at 500 μM). Complete inhibition of contraction occurred without inhibition of increases in intracellular calcium concentrations ([Ca 2+ ] i ).

Background Papaverine has been used to combat vasospasm in human saphenous veins (HSV) as an alternative to high pressure distension techniques. Papaverine acts as a nonspecific inhibitor of phosphodiesterases, increasing both cGMP and cAMP. We hypothesized that the mechanism by which papaverine reduces force through regulation of actin depolymerization and myosin light chain dephosphorylation. Methods HSV was pretreated with 1 mM papaverine followed by 5 μM norepinephrine. Force and intracellular [Ca 2+ ] i levels were concurrently measured using a Fluoroplex. Tissue was snap frozen for biochemical analyses. Results Papaverine completely inhibited norepinephrine-induced force development and intracellular [Ca 2+ ] i . Papaverine pretreatment led to increases in the phosphorylation of heat shock-related protein 20 (HSPB6), dephosphorylation of myosin light chain, and depolymerization of actin. Pretreament of papaverine led to an association of HSPB6 with 14-3-3 eta as determined by immunoprecipitation and mass spectrometry analysis. The interaction of HSPB6 with 14-3-3 leads to dissociation and activation of cofilin, an actin depolymerizing protein. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves decreases in intracellular [Ca 2+ ] i by inhibiting the calcium regulatory pathways, leading to the dephosphorylation of myosin light chain. Additionally papaverine induces the phosphorylation of HSPB6, which leads to depolymerization of actin. Papaverine inhibits HSV spasm via multiple molecular mechanisms.

Objective The human saphenous vein (HSV) is the most effective conduit for infrainguinal peripheral bypass grafting. Optimal vein graft preparation is critical to vein graft patency. Graft marking using surgical skin markers is detrimental to endothelial and smooth muscle function of the graft. The objective of this study was to evaluate the effect of FD&C Brilliant Blue No. 1 (FCF) as an alternative for vein graft marking. Methods Segments of HSV were collected after typical harvest and graft preparation. The tissues were cut into rings and either left unmarked or marked with FCF (2.6 mM). The functional viability of the rings was determined in a muscle bath. Results Marking with FCF did not impair contractility of the smooth muscle (Figure 1A). Contractility was restored in HSV segments that had no functional contractile responses after harvest and preparation (Figure 1B). To determine the mechanism of this protective effect of FCF, purinergic receptors were activated with BzATP with and without pre-incubation with FCF (50μM) or a purinergic receptor antagonist (oATP, control) and the rise in [Ca 2+ ] i was measured. FCF and oATP reduced the BZATP-evoked rise in [Ca 2+ ] i (Figure 1C), suggesting that the salutary effects of FCF were due to inhibition of a P2X or P2Y receptor. Conclusions FCF is a non-toxic alternative to surgical skin markers that antagonizes purinergic receptors in HSV. FCF restored functional viability to injured vascular smooth muscle. FCF may protect HSV against injury-elicited ‘danger’ signals such as extracellular ATP that is released during injury-induced preparation.