Off-the-Shelf Synthetic Biodegradable Grafts Transform In Situ into a Living Arteriovenous Fistula in a Large Animal Model.

Current vascular access options require frequent interventions. In situ tissue engineering (TE) may overcome these limitations by combining the initial success of synthetic grafts with long-term advantages of autologous vessels by using biodegradable grafts that transform into autologous vascular tissue at the site of implantation. Scaffolds (6 mm-Ø) made of supramolecular polycarbonate-bisurea (PC-BU), with a polycaprolactone (PCL) anti-kinking-coil, are implanted between the carotid artery and jugular vein in goats. A subset is bio-functionalized using bisurea-modified-Stromal cell-derived factor-1α (SDF1α) derived peptides and ePTFE grafts as controls. Grafts are explanted after 1 and 3 months, and evaluated for material degradation, tissue formation, compliance, and patency. At 3 months, the scaffold is resorbed and replaced by vascular neo-tissue, including elastin, contractile markers, and endothelial lining. No dilations, ruptures, or aneurysms are observed and grafts are successfully cannulated at termination. SDF-1α-peptide-biofunctionalization does not influence outcomes. Patency is lower in TE grafts (50%) compared to controls (100% patency), predominantly caused by intimal hyperplasia. Rapid remodeling of a synthetic, biodegradable vascular scaffold into a living, compliant arteriovenous fistula is demonstrated in a large animal model. Despite lower patency compared to ePTFE, transformation into autologous and compliant living tissue with self-healing capacity may have long-term advantages.

The effect of chronic kidney disease on tissue formation of in situ tissue-engineered vascular grafts.

Vascular in situ tissue engineering encompasses a single-step approach with a wide adaptive potential and true off-the-shelf availability for vascular grafts. However, a synchronized balance between breakdown of the scaffold material and neo-tissue formation is essential. Chronic kidney disease (CKD) may influence this balance, lowering the usability of these grafts for vascular access in end-stage CKD patients on dialysis. We aimed to investigate the effects of CKD on in vivo scaffold breakdown and tissue formation in grafts made of electrospun, modular, supramolecular polycarbonate with ureido-pyrimidinone moieties (PC-UPy). We implanted PC-UPy aortic interposition grafts (n = 40) in a rat 5/6th nephrectomy model that mimics systemic conditions in human CKD patients. We studied patency, mechanical stability, extracellular matrix (ECM) components, total cellularity, vascular tissue formation, and vascular calcification in CKD and healthy rats at 2, 4, 8, and 12 weeks post-implantation. Our study shows successful in vivo application of a slow-degrading small-diameter vascular graft that supports adequate in situ vascular tissue formation. Despite systemic inflammation associated with CKD, no influence of CKD on patency (Sham: 95% vs CKD: 100%), mechanical stability, ECM formation (Sirius red+, Sham 16.5% vs CKD 25.0%-p:0.83), tissue composition, and immune cell infiltration was found. We did find a limited increase in vascular calcification at 12 weeks (Sham 0.08% vs CKD 0.80%-p:0.02) in grafts implanted in CKD animals. However, this was not associated with increased stiffness in the explants. Our findings suggest that disease-specific graft design may not be necessary for use in CKD patients on dialysis.

A plasma creatinine- and urea-based equation to estimate glomerular filtration rate in rats.

Monitoring renal function is a vital part of kidney research involving rats. The laborious measurement of glomerular filtration rate (GFR) with administration of exogenous filtration markers does not easily allow serial measurements. Using an in-house database of inulin clearances, we developed and validated a plasma creatinine- and plasma urea-based equation to estimate GFR in a large cohort of male rats [development cohort n = 325, R2 = 0.816, percentage of predictions that fell within 30% of the true value (P30) = 76%] that had high accuracy in the validation cohort (n = 116 rats, R2 = 0.935, P30 = 79%). The equation was less accurate in rats with nonsteady-state creatinine, in which the equation should therefore not be used. In conclusion, applying this equation facilitates easy and repeatable estimates of GFR in rats.NEW & NOTEWORTHY This is the first equation, that we know of, which estimates glomerular filtration rate in rats based on a single measurement of body weight, plasma creatinine, and plasma urea.

Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for "acquired" proteins or micro RNAs (miRNAs) repressing "resident" mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.

Endothelial progenitor cell levels in obese men with the metabolic syndrome and the effect of simvastatin monotherapy vs. simvastatin/ezetimibe combination therapy.

AIMS: Endothelial progenitor cells (EPCs) contribute to endothelial regeneration and thereby protect against cardiovascular disease (CVD). Patients with manifest CVD have reduced EPC levels, but it is not clear if this also occurs in subjects at high CVD risk without manifest atherosclerotic disease. Therefore, we aimed to first, measure circulating levels of EPCs in subjects without manifest CVD but at high cardiovascular risk due to obesity and presence of the metabolic syndrome. Second, we evaluated the effect on EPC levels of two lipid-lowering treatments. METHODS AND RESULTS: Circulating CD34+KDR+ EPC levels were reduced by nearly 40% in obese men with the metabolic syndrome compared to non-obese healthy controls (331 +/- 193 vs. 543 +/- 164 EPC/mL, P = 0.006). In a randomized double-blind cross-over study comparing intensive lipid-lowering treatment using 80 mg simvastatin mono-treatment with combination treatment of 10 mg simvastatin and 10 mg ezetimibe, we found a similar treatment effect on EPC levels. Secondary analyses of these data suggested that both treatment regimens had increased circulating EPCs to control levels (626 +/- 428 after combination treatment, P < 0.01; 524 +/- 372 EPC/mL after monotherapy, P < 0.05). Serum levels of EPC-mobilizing factor SCF-sR correlated with reduced EPC levels and normalized concurrently with treatment. CONCLUSION: EPC levels are reduced in apparently healthy men with abdominal obesity and the metabolic syndrome, even in the absence of manifest CVD. This is important as EPCs contribute to endothelial regeneration and thereby protect against CVD. SCF-sR may be a candidate serum marker of circulating EPC levels. Treatment with low-dose statin with ezetimibe combination therapy or high-dose statin monotherapy has similar effects on the reduced EPC levels.

End-stage renal disease causes an imbalance between endothelial and smooth muscle progenitor cells.

Patients with end-stage renal disease (ESRD) on hemodialysis have an increased risk of cardiovascular disease (CVD). Circulating endothelial progenitor cells (EPC) contribute to vascular regeneration and repair, thereby protecting against CVD. However, circulating smooth muscle progenitor cells (SPC) may contribute to adverse vascular remodeling. We hypothesized that an imbalance occurs between EPC and SPC in ESRD patients and sampled progenitor cells from 45 ESRD patients receiving regular treatment. Our study is the first to show reduced numbers of CD34+KDR+ hematopoietic stem cell (HSC)-derived EPC (type I EPC). Furthermore, monocyte-derived EPC cultured from mononuclear cells (type II EPC) were reduced in number and had a reduced capacity to stimulate endothelial cell angiogenesis. In contrast, SPC outgrowth was unaffected. In vitro incubation with uremic serum impaired type II EPC outgrowth from healthy donor mononuclear cells and did not influence SPC outgrowth. The hemodialysis procedure itself induced HSC apoptosis and caused an acute depletion of circulating EPC. Taken together, the decreased number and impaired function of EPC are compatible with impaired endogenous vascular repair in hemodialysis patients, whereas the unaffected SPC numbers suggest that the potential of progenitor cells to contribute to adverse remodeling is retained. This EPC-SPC imbalance may contribute to the acceleration of CVD in ESRD patients and could offer novel therapeutic targets.

Involvement of the proteasome in activation of endothelial nitric oxide synthase.

Nitric oxide originating from the endothelial cells of the vessel wall is essential for the vascular system. It is produced by the enzyme endothelial nitric oxide synthase (eNOS). Cellular eNOS activity is affected by changes in eNOS synthesis. To address whether degradation also contributes to eNOS activity, the effect of proteasome inhibitors on eNOS-mediated NO synthesis was studied in the microvascular endothelial cell line bEnd.3 and in cultured primary aortic endothelial cells. Surprisingly, agonist-induced increases in eNOS activity were reduced to 42 and 50% in the presence of the proteasome inhibiting drugs MG132 and clasto-lactacystin-beta-lactone, respectively (P < 0.01). The decrease in activity occurred within 1 hour of drug treatment and was not accompanied by a change in intracellular levels of either eNOS or its inhibitor caveolin-1. Taken together, these data may indicate that eNOS is regulated by an interacting protein, different from caveolin-1, that inhibits its activity and is rapidly degraded by the proteasome in the presence of eNOS agonists.

Endothelial nitric oxide synthase activity is linked to its presence at cell-cell contacts.

The enzyme endothelial nitric oxide synthase (eNOS) is essential for vascular integrity. Many studies have demonstrated a link between the localization and activity of eNOS. Here, we studied the influence of cell-cell contact on this link in the microvascular endothelial bEnd.3 cell line. By immunofluorescence microscopy, eNOS localization at the plasma membrane was found to be dependent on cell-cell contact. In particular, eNOS was highly enriched at the intercellular contact sites. Further analysis showed that the pattern of eNOS localization at the plasma membrane resembled that of PECAM-1 (platelet endothelial cell adhesion molecule 1), but not that of the adherens junction proteins VE (vascular endothelial)-cadherin and plakoglobin. eNOS that was localized at the contact sites was, in part, Triton X-100-insoluble, in contrast with eNOS at the Golgi complex, which may indicate an association of eNOS with the actin cytoskeleton. Interestingly, eNOS activity was up-regulated in confluent monolayers compared with subconfluent cells, while there was no difference in eNOS expression. This correlation between cell confluence and eNOS activity was also found when primary bovine aortic endothelial cells were studied. These data imply that cell-cell contact induces the localization of eNOS at intercellular junctions, which is required for agonist-induced eNOS activation.