Coronary artery disease (CAD) is the single leading cause of death worldwide. Advances in treatment and management have significantly improved patient outcomes. On the other hand, although mortality rates have decreased, more people are left with sequelae that require additional treatment and hospitalization. Moreover, patients with severe nonrevascularizable CAD remain with only the option of heart transplantation, which is limited by the shortage of suitable donors. In recent years, cell-based regenerative therapy has emerged as a possible alternative treatment, with several regenerative medicinal products already in the clinical phase of development and others emerging as competitive preclinical solutions. Recent evidence indicates that pericytes, the mural cells of blood microvessels, represent a promising therapeutic candidate. Pericytes are abundant in the human body, play an active role in angiogenesis, vessel stabilization and blood flow regulation, and possess the capacity to differentiate into multiple cells of the mesenchymal lineage. Moreover, early studies suggest a robustness to hypoxic insult, making them uniquely equipped to withstand the ischemic microenvironment. This review summarizes the rationale behind pericyte-based cell therapy and the progress that has been made toward its clinical application. We present the different sources of pericytes and the case for harvesting them from tissue leftovers of cardiovascular surgery. We also discuss the healing potential of pericytes in preclinical animal models of myocardial ischemia (MI) and current practices to upgrade the production protocol for translation to the clinic. Standardization of these procedures is of utmost importance, as lack of uniformity in cell manufacturing may influence clinical outcome.

The cytochrome P450s (CYPs) represent a highly divergent class of enzymes involved in the oxidation of organic compounds. A subgroup of CYPs metabolize ω3-arachidonic and linoleic acids and ω6-docosahexaenoic and eicosapentaenoic polyunsaturated fatty acids (PUFAs) into a series of related biologically active mediators. Over the past 20 years, increasing evidence has emerged for a role of these PUFA-derived mediators in physiological and pathophysiological processes in the vasculature, during inflammation, and in the regulation of metabolism. With recent technological advances and increased availability of lipid mass spectroscopy, we are now starting to discern the patterns of these CYP-PUFA products in health and disease. These analyses not only are revealing the diverse spectrum of lipid nutrients regulated by CYPs, but also clearly indicate that the balance of these mediators changes with dietary intake of different PUFA classes. These findings suggest that we are only just beginning to understand all of the relevant lipid species produced by CYP pathways. Moreover, we are still a long way from understanding the nature and presence of their receptors, their tissue expression, and the pathophysiological processes they regulate. This review highlights these future issues in the context of lipid-metabolizing CYP enzymes, focusing particularly on the CYP450 family of epoxygenases and the lipid mediators they produce.

Aims Homozygosity for a four-missense single-nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this longevity-associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. Methods and results Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild-type-BPIFB4 exerted no benefit. Interestingly, LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and up-regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 up-regulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. Conclusions In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm2) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. Geltrex™ basement matrix at 5 μl/cm2 in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

Peroxisome proliferator activated receptor β/δ (PPARβ/δ) has pro-angiogenic functions, but whether PPARβ/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARβ/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARβ/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARβ/δ- and sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARβ/δ in the endothelium and support the targeting of PPARβ/δ in regulating EC behaviour and boosting tissue maintenance and repair.