Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum and can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study a re-defined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding 5 glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid over the course of restenosis progression to investigate potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization.

Drug-eluting stents are the most commonly employed method to control post-angioplasty restenosis. Unfortunately, they exacerbate life-threatening stent thrombosis because of endothelium damage caused by both drug and stenting. To solve this major medical problem, an endothelium-protective and stent-free anti-restenotic method is highly desirable. Here we have generated a biomimetic intravenous delivery system using dendritic polymer-based nanoclusters, which were coated with platelet membranes for targeting to the injured arterial wall where restenosis occurs. These nanoclusters were loaded with an endothelium-protective epigenetic inhibitor (JQ1) or an endothelium-toxic status quo drug (rapamycin), and compared for their ability to mitigate restenosis without hindering the process of re-endothelialization. Fluorescence imaging of Cy5-tagged biomimetic nanoclusters indicated their robust homing to injured, but not uninjured arteries. Two weeks after angioplasty, compared to no-drug control, both rapamycin- and JQ1-loaded biomimetic nanoclusters substantially reduced (by >60%) neointimal hyperplasia, the primary cause of restenosis. However, whereas the rapamycin formulation impaired the endothelial re-coverage of the denuded inner arterial wall, the JQ1 formulation preserved endothelial recovery. In summary, we have created an endothelium-protective anti-restenotic system with biomimetic nanoclusters containing an epigenetic inhibitor. This system warrants further development for a non-thrombogenic and stent-free method for clinical applications.

Objectives: This study intended to confirm whether skin perfusion pressure (SPP) could predict the outcome of ischemic wound healing. Patients and methods: Sixty-two limbs in 53 patients with conservative therapy were enrolled in this study. A SPP value of 40 mmHg was adopted as the criterion for making clinical decisions. The outcome one month after SPP measurement was classified as "improved" (diameter of ulcer decreased ≥ 20% or demarcation of gangrene became well defined) or "no change or worse" (others), and the fate of wound was classified as "healed" or "not healed". The evaluated influential factors on the outcome at one month included age, sex, presence of arteriosclerosis obliterans, collagen disease, hypertension, diabetes mellitus, hemodialysis, wound infection, wound management, and SPP ≥ 40 mmHg. Results: Using a criterion of SPP ≥ 40 mmHg, the outcome at one month could be predicted with a sensitivity: of 75.0%, a specificity: of 82.6%, and an accuracy: of 80.6%. The receiver operating characteristic curve indicated our criterion to be appropriate. Logistic regression analysis showed SPP ≥ 40 mmHg to be an independent factor (P < 0.0001) with the odds ratio of 14.2 (95% CI 3.6-55.8). Conclusions: SPP, using a cutoff value of 40 mmHg, can predict the ischemic wound healing with conservative therapy.

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SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

BackgroundDrug-eluting stents (DES) represent the main-stream management of restenosis following treatments of occlusive cardiovascular diseases. However, DES cannot eliminate instent restenosis yet exacerbate thrombogenic risks. To achieve dual inhibition of restenotic smooth muscle cell (SMC) de-differentiation/proliferation and thrombogenic endothelial cell (EC) dysfunction, a common target in both cell types, has been long-sought after. We evaluated the potential of protein kinase RNA-like endoplasmic reticulum kinase (PERK) as such a target for low-thrombogenic anti-restenotic intervention.Methods and ResultsWe used a rat angioplasty model of restenosis and a FeCl3-induced mouse model of thrombosis. Loss-or gain-of-function was achieved by PERK inhibition (GSK2606414, siRNA) or overexpression (adenovirus). Restenosis was robustly mitigated by GSK2606414 administered either via injected (i.v.) lesion-homing platelet membrane-coated nanoclusters or a perivascular hydrogel; it was enhanced by PERK transgene. Whereas PERK inhibition blocked, its overexpression exacerbated PDGF-induced human aortic SMC de-differentiation (reduced smooth muscle α-actin or αSMA) and proliferation (BrdU incorporation). Further, PERK activity promoted STAT3 activation but inhibited SRF transcriptional (luciferase) activity; its protein co-immunoprecipitated with STAT3 and also MRTF-A, the SRF activator for αSMA transcription. Importantly, PERK inhibition also prevented TNFα-induced impairment of human EC growth and upregulation of thrombogenic tissue factor, both in vitro and ex vivo. In vivo, oral gavage of GSK2606414 preserved ~50% of the normal blood flow 60 min after FeCl3-induced vascular injury.ConclusionsPERK inhibition is dual beneficial in mitigating restenosis and thrombosis, thus implicating a potential design for anti-restenotic intervention to overcome the thrombogenicity of DES.

The basic structure of adventitial collagen fiber was maintained in abdominal aortas regardless of location or age. Because the molecular structure at the subfibril level changed at abdominal aorta and enhanced in aortas with AAA, alterations in the molecular structure of adventitial collagen might be associated with aneurysmal formation.