Bioresorbable zinc stent with ultra-thin center struts attenuates stent jail in porcine femoral artery bifurcations.

INTRODUCTION: An ultra-thin, fracture-resistant and bioresorbable stent may be advantageous for provisional stenting in vessel bifurcations, if catheter passage and side-branch post-dilatation is facilitated to prevent a 'stent jail' by struts obstructing the orifice of a major side branch. MATERIAL AND METHODS: We studied a highly radiopaque, slowly bioresorbable zinc alloy stent characterized by a novel design of a radiopaque-marked region of ultra-thin struts in the center of the stent. The stent is characterized by an extended range flexibility and high fracture resistance. Zn-stents and Zn-drug eluting stents (DES) were implanted opposite to rigid Nitinol stents into both femoral artery bifurcations of 21 juvenile pigs, followed for one and three months and studied by angiography and histomorphometry.Results and conclusion: Bare Zn-stents with thinner stent struts showed less neointimal hyperplasia compared to Zn-stents with thicker struts. Neointimal formation was further reduced by 12% in Zn-alloy DES. Both, bare Zn-stents and Zn-DES, can be precisely positioned into the femoral artery bifurcation, allowing easy balloon catheter passage through the very thin strut mesh. Side branch orifices remained open after Zn-stent deployment without stent jailing. No stent fractures or particles emboli occurred after the deployment. A Zn-stent with ultra-thin center struts may be useful for provisional stenting in vessel bifurcations.

GDF-15 Deficiency Reduces Autophagic Activity in Human Macrophages In Vitro and Decreases p62-Accumulation in Atherosclerotic Lesions in Mice.

(1) Background: Growth differentiation factor-15 (GDF-15) is associated with cardiovascular diseases and autophagy in human macrophages (MΦ). Thus, we are interested in investigating autophagic mechanisms with special respect to the role of GDF-15. (2) Methods: Recombinant (r)GDF-15 and siRNA GDF-15 were used to investigate the effects of GDF-15 on autophagic and lysosomal activity, as well as autophagosome formation by transmission electron microscopy (TEM) in MΦ. To ascertain the effects of GDF-15-/- on the progression of atherosclerotic lesions, we used GDF-15-/-/ApoE-/- and ApoE-/- mice under a cholesterol-enriched diet (CED). Body weight, body mass index (BMI), blood lipid levels and lumen stenosis in the brachiocephalic trunk (BT) were analyzed. Identification of different cell types and localization of autophagy-relevant proteins in atherosclerotic plaques were performed by immunofluorescence. (3) Results: siGDF-15 reduced and, conversely, rGDF-15 increased the autophagic activity in MΦ, whereas lysosomal activity was unaffected. Autophagic degradation after starvation and rGDF-15 treatment was observed by TEM. GDF-15-/-/ApoE-/- mice, after CED, showed reduced lumen stenosis in the BT, while body weight, BMI and triglycerides were increased compared with ApoE-/- mice. GDF-15-/- decreased p62-accumulation in atherosclerotic lesions, especially in endothelial cells (ECs). (4) Conclusion: GDF-15 seems to be an important factor in the regulation of autophagy, especially in ECs of atherosclerotic lesions, indicating its crucial pathophysiological function during atherosclerosis development.

PAC1 deficiency attenuates progression of atherosclerosis in ApoE deficient mice under cholesterol-enriched diet.

The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is vasoactive and cytoprotective and exerts immunoregulatory functions throughout the nervous, neuroendocrine cardiovascular and immune systems in health and disease. PACAP mainly acts through PAC1 receptor signaling in neuronal communication, but the role of PAC1 in immune regulation of atherosclerosis is not known. Here, we generated PAC1-/-/ApoE-/- mice to test, whether PAC1-/- influences plasma cholesterol-/triglyceride levels and/or atherogenesis in the brachiocephalic trunk (BT) seen in ApoE-/- mice, under standard chow (SC) or cholesterol-enriched diet (CED). Furthermore, the effect of PAC1-/-, on inflammatory, autophagy-, apoptosis- and necroptosis-relevant proteins in atherosclerotic plaques was determined. In plaques of PAC1-/-/ApoE-/- mice fed a SC, the immunoreactivity for apoptotic, autophagic, necroptotic and proinflammatory proteins was increased, however, proliferation was unaffected. Interestingly, without affecting hyperlipidemia, PAC1-/- in ApoE-/- mice remarkably reduced CED-induced lumen stenosis seen in ApoE-/- mice. Thus, PAC1-/- allows unchecked inflammation, necroptosis and decreased proliferation during SC, apparently priming the BT to develop reduced atheroma under subsequent CED. Remarkably, no differences in inflammation/necroptosis signatures in the atheroma under CED between PAC1-/-/ApoE-/- and ApoE-/- mice were observed. These data indicate that selective PAC1 antagonists should offer potential as a novel class of atheroprotective therapeutics, especially during hypercholesterolemia.

NOD1 and NOD2 of the innate immune system is differently expressed in human clear cell renal cell carcinoma, corresponding healthy renal tissue, its vasculature and primary isolated renal tubular epithelial cells.

PURPOSE: NOD1 and NOD2 (nucleotide-binding oligomerization domain)-receptors are intracellular receptors and belong to the family of pattern recognition receptors being present in both human and murine renal tubular cells. Besides, NOD1 has been proved to promote apoptosis, upon its overexpression. Hence, we aimed to investigate NOD1 and NOD2 expression in human clear cell renal cell carcinoma (ccRCC). METHODS: Tumor and corresponding adjacent healthy tissues from 41 patients with histopathological diagnosis of ccRCC as well as primary isolated renal tubular epithelial cells (TECs) and tumor tissue from a murine xenograft model using CAKI-1 ccRCC cells were analyzed. RESULTS: NOD1 and NOD2 mRNA was constitutively expressed in both tumor and adjacent healthy renal tissue, with NOD1 being significantly lower and in contrast NOD2 significantly higher expressed in tumor tissue compared to healthy tissues. Immunohistochemically, NOD1 was located not only in the cytoplasm, but also in the nucleus in ccRCC tissue whereas NOD2 was solely localized in the cytoplasm in both human ccRCC as well as in the healthy tubular system. Focusing on the vasculature, NOD2 displayed broader expression than NOD1. In primary TECs as well as CAKI-1 cells NOD1 and NOD2 was constitutively expressed and increasable upon LPS stimulation. In the mouse xenograft model, human NOD1 mRNA was significantly higher expressed compared to NOD2. In contrast hereto, we observed a shift towards lower mouse NOD1 compared to NOD2 mRNA expression. CONCLUSION: In view of reduced apoptosis-associated NOD1 expression in ccRCC tissue opposed to higher expression of NOD2 in tumor vasculature, inducibility of NOD expression in TECs as well as the detected shift of NOD1 and NOD2 expression in the mouse xenograft model, modulation of NOD receptors might, therefore, provide a molecular therapeutic approach in ccRCC.

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP-/-) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP-/-/ApoE-/- mice showed an increased lumen stenosis compared to ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice, the immunoreactive areas of TNF+, IL-1ß+, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE-/- under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP-/-/ApoE-/- and ApoE-/- mice under the respective feeding regime. Thus, PACAP-/-/ApoE-/- mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.

Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis.

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease.