The SYNERGY biodegradable polymer everolimus eluting coronary stent: Porcine vascular compatibility and polymer safety study.

AIMS: SYNERGY is a novel platinum chromium alloy stent that delivers abluminal everolimus from an ultrathin poly-lactide-co-glycide (PLGA) biodegradable polymer. This study evaluated the in vivo degradation of the polymer coating, everolimus release time course, and vascular compatibility of the SYNERGY stent. METHODS AND RESULTS: SYNERGY stents were implanted in arteries of domestic swine. Devices were explanted at predetermined time points (up to 120 days) and the extent of PLGA coating or everolimus remaining on the stents was quantified. Everolimus levels in the arterial tissue were also evaluated. A pathological analysis on coronary arteries of single and overlapping stents was performed at time points between 5 and 270 days. PLGA bioabsorption began immediately after implantation, and drug release was essentially complete by 90 days; PLGA absorption was substantially complete by 120 days (>90% of polymer was absorbed) leaving a bare metal SYNERGY stent. Vascular response was similar among SYNERGY and control stents (bare metal, polymer-only, and 3× polymer-only). Mild increases in para-strut fibrin were seen for SYNERGY at an early time point with no significant differences in all other morphological and morphometric parameters through 270 days or endothelial function (eNOS immunostaining) at 90 or 180 days. Inflammation was predominantly minimal to mild for all device types. CONCLUSION: In a swine model, everolimus was released by 90 days and PLGA bioabsorption was complete shortly thereafter. The SYNERGY stent and its biodegradable polymer, even at a 3× safety margin, demonstrated vascular compatibility similar to bare metal stent controls.

Bipolar multi-electrode balloon catheter radiofrequency renal denervation with the Vessix system: preclinical safety evaluation.

AIMS: A bipolar multi-electrode 7 Fr-compatible balloon-catheter radiofrequency (RF) renal denervation system (Vessix™ Renal Denervation System; Boston Scientific, Marlborough, MA, USA) was evaluated for safety in domestic swine. METHODS AND RESULTS: Renal arteries of 27 swine received overlapping treatments proximally/single treatments distally to mimic balloon overlap clinically. Each histopathology cohort (30, 90, 180 days) had four RF-treated and three sham-treated (no RF energy delivered) animals, with the response of artery/surrounding nerves to bilateral treatment examined (42 arteries). Scanning electron microscopy of the renal artery flow surface for endothelialisation was performed in six additional pigs (three at each of 30 and 90 days: 12 arteries) following unilateral whole artery treatment with proximal overlap: RF one side, sham the other side. Power was ~1 watt, treatment duration 30 seconds, target temperature 68°C. Renal histology and assessment for off-target injury was performed in all 27 swine. Renal artery thermal injury was transmural and segmental involving <10% to >90% of the circumference (typically 30-60%) with segmental neointimal hyperplasia exceeding shams but haemodynamically trivial (maximum stenosis 17.7%). Healing of necrotic arterial media was by replacement fibrosis. Overlying nerves also became fibrotic. Endothelialisation was focally incomplete at 30 days but confluent at 90 days. No off-target injury occurred outside the renal arteries. CONCLUSIONS: Safety was demonstrated.

Matrix metalloproteinase 9 contributes to gut microbe homeostasis in a model of infectious colitis.

BACKGROUND: Inflammatory bowel diseases are associated with increased expression of zinc-dependent Matrix Metalloproteinase 9 (MMP-9). A stark dysregulation of intestinal mucosal homeostasis has been observed in patients with chronic inflammatory bowel diseases. We therefore sought to determine the contribution of MMP-9 to the pathogenesis of Citrobacter rodentium-induced colitis and its effects on gut microbiome homeostasis. RESULTS: Wild-type and MMP-9-/- mice aged 5-6 weeks were challenged with C. rodentium by orogastric gavage and sacrificed either 10 or 30 days post-infection. Disease severity was assessed by histological analysis of colonic epithelial hyperplasia and by using an in vivo intestinal permeability assay. Changes in the inflammatory responses were measured by using qPCR, and the composition of the fecal microbiome evaluated with both qPCR and terminal restriction fragment length polymorphism. Activation and localization of MMP-9 to the apical surface of the colonic epithelium in response to C. rodentium infection was demonstrated by both zymography and immunocytochemistry. The pro-inflammatory response to infection, including colonic epithelial cell hyperplasia and barrier dysfunction, was similar, irrespective of genotype. Nonmetric multidimensional scaling of terminal restriction fragments revealed a different fecal microbiome composition and C. rodentium colonization pattern between genotypes, with MMP-9-/- having elevated levels of protective segmented filamentous bacteria and interleukin-17, and lower levels of C. rodentium. MMP-9-/- but not wild-type mice were also protected from reductions in fecal microbial diversity in response to the bacterial enteric infection. CONCLUSIONS: These results demonstrate that MMP-9 expression in the colon causes alterations in the fecal microbiome and has an impact on the pathogenesis of bacterial-induced colitis in mice.

Podocyte-specific loss of Cdc42 leads to congenital nephropathy.

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.

Expression patterns of ShcD and Shc family adaptor proteins during mouse embryonic development.

The Src homology and collagen (Shc) proteins function as molecular adaptors in signaling pathways mediated by a variety of cell surface receptors. Of the four mammalian Shc proteins, ShcD is the least characterized. To this end, ShcD expression was documented and compared to that of other Shc family proteins. In the developing mouse embryo, expression of ShcD overlaps with that of other Shc proteins in the central nervous system, with specific distribution in post-mitotic neurons. In addition, robust ShcD expression is seen within differentiated epithelial cells of several organs, as well as in skeletal and cardiac muscle, and various tissues of neural crest origin. Interestingly, all Shc family members are expressed in hypertrophic chondrocytes, the first report of Shc protein expression in the developing skeleton. The unique tissue distribution patterns of Shc proteins likely contribute to their complex tissue-specific signaling functions during embryogenesis.

Adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.