Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the body\u27s largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease

Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α

Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart

Acute visual loss and chorioretinal infarctionafter photodynamic therapy combined withintravitreal triamcinolone

.Abstract PURPOSE: To report acute visual loss associated with dynamic vascular changes after photodynamic therapy (PDT) combined with intravitreal triamcinolone (IVTA) for the treatment of occult choroidal neovascularization (CNV). METHODS: An 86-year-old woman complained of visual loss in her left eye. Angiographic examination showed a serous pigment epithelium detachment complicated by CNV. She underwent combined treatment with IVTA (4 mg) followed by standard verteporfin PDT administered after a 5-day interval. RESULTS: The patient developed vision loss 1 day after PDT. Ophthalmoscopic examination disclosed an acute serous neurosensory retinal detachment. Fluorescein angiography showed a large area of early hypofluorescence in correspondence to and extending beyond the photodynamic spot. Neurosensory retinal vessels involvement with dilation of the retinal arterioles and capillary nonperfusion were also revealed. Indocyanine green angiography showed choroidal infarction within the collateral choroid included in the area of light exposure, with associated nonperfusion of medium and large choroidal vessels being revealed. Five days after PDT, spontaneous severe bleeding with breakthrough into the vitreous occurred, in addition to an RPE tear. CONCLUSIONS: Acute loss of vision associated with vascular changes in retinal and choroidal circulation represents an uncommon but serious complication following combined PDT and IVTA. These risks should be carefully considered in combination therapies

HIV-1-mediated delivery of a short hairpin RNA targeting vascular endothelial growth factor in human retinal pigment epithelium cells

Background: Vascular endothelial growth factor (VEGF) has been shown to play a major role in the pathological neovascularisation that occurs in degenerative retinal diseases like age-related macular degeneration (AMD). Although several approaches to attenuate VEGF show significant promise, repeated treatments are required to achieve therapeutic benefits. As lentiviruses efficiently and stably infect resting cells, a human immunodeficiency virus type 1 (HIV-1)-based vector was used for the delivery and long-term endogenous expression of a short hairpin RNA (shRNA) specific for VEGF in postmitotic human retinal pigment epithelium (RPE) cells. Methods: An HIV-1 vector expressing a shRNA targeting VEGF was developed and adopted to transduce RPE cell cultures, in both normoxic and hypoxic conditions in vitro. Intracellular VEGF expression was analysed by western blotting, and the release of VEGF in culture supernatants was determined by ELISA. Results: At least 90% of RPE cells were successfully transduced by HIV-1 virions. Inhibition of VEGF expression and reduction by 95% of VEGF release in transduced cells were achieved. Moreover, shRNA-VEGF effectively and specifically prevented hypoxia-induced VEGF upregulation. Conclusion: HIV-1-mediated delivery of a shRNA-VEGF leading to gene expression knockdown could represent a novel therapeutic strategy against neovascularisation-related eye diseases

Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea.

Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder causedby mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence ofunsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visualimpairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressedand exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of theKSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in culturedprimary keratinocytes (n¼3) and keratocytes (n¼4). Immunohistochemistry, semiquantitative RT-PCR, insitu RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneasKS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium.A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. Asexpected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues ontoKSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and inthe endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytesexpress and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarlyexpression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromalkeratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and expressvery little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare geneticdisorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targetedtherapeutic treatments of the ocular features in MCD patients

MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs

Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition(1), particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue(2). Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled