An assessment of prevalence of Type 1 CFI rare variants in European AMD, and why lack of broader genetic data hinders development of new treatments and healthcare access.

PURPOSE: Advanced age-related macular degeneration (AAMD) risk is associated with rare complement Factor I (FI) genetic variants associated with low FI protein levels (termed 'Type 1'), but it is unclear how variant prevalences differ between AMD patients from different ethnicities. METHODS: Collective prevalence of Type 1 CFI rare variant genotypes were examined in four European AAMD datasets. Collective minor allele frequencies (MAFs) were sourced from the natural history study SCOPE, the UK Biobank, the International AMD Genomics Consortium (IAMDGC), and the Finnish Biobank Cooperative (FINBB), and compared to paired control MAFs or background population prevalence rates from the Genome Aggregation Database (gnomAD). Due to a lack of available genetic data in non-European AAMD, power calculations were undertaken to estimate the AAMD population sizes required to identify statistically significant association between Type 1 CFI rare variants and disease risk in different ethnicities, using gnomAD populations as controls. RESULTS: Type 1 CFI rare variants were enriched in all European AAMD cohorts, with odds ratios (ORs) ranging between 3.1 and 7.8, and a greater enrichment was observed in dry AMD from FINBB (OR 8.9, 95% CI 1.49-53.31). The lack of available non-European AAMD datasets prevented us exploring this relationship more globally, however a statistical association may be detectable by future sequencing studies that sample approximately 2,000 AAMD individuals from Ashkenazi Jewish and Latino/Admixed American ethnicities. CONCLUSIONS: The relationship between Type 1 CFI rare variants increasing odds of AAMD are well established in Europeans, however the lack of broader genetic data in AAMD has adverse implications for clinical development and future commercialisation strategies of targeted FI therapies in AAMD. These findings emphasise the importance of generating more diverse genetic data in AAMD to improve equity of access to new treatments and address the bias in health care.

Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin II-induced hypertension in rats.

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine 105 (S105) for the actions of GATA4 in an angiotensin II (AngII)-induced hypertension rat model. Adenoviral constructs overexpressing wild-type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg/kg/h) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT-qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII-induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult-to-foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild-type or mutated GATA4. Our results indicate that GATA4 reduces AngII-induced responses by interfering with pro-fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.

Transcription factor PEX1 modulates extracellular matrix turnover through regulation of MMP-9 expression.

The phenylephrine-induced complex-1 (PEX1) transcription factor, also known as zinc-finger protein 260 (Zfp260), is an effector of endothelin-1 and α1-adrenergic signaling in cardiac hypertrophy. However, the role of PEX1 in transcriptional regulation of myocardial remodeling remains largely unknown. In the present study, we used PEX1 gain- and loss-of-function to examine the effects of PEX1 on left ventricular remodeling. Adenoviral constructs expressing PEX1, antisense PEX1, or LacZ were delivered by local injection into the anterior wall of the left ventricle in Sprague-Dawley rats. PEX1 overexpression led to induction of hypertrophic gene program and increased fibrosis. In agreement with this, the expression of genes involved in the fibrotic process, such as collagens I and III, matrix metalloproteinases (MMPs), fibronectin-1, transforming growth factor beta-1 and connective tissue growth factor, were significantly up-regulated following PEX1 overexpression, whereas silencing of PEX1 significantly inhibited the expression of pro-fibrotic genes and increased left ventricular ejection fraction and fractional shortening. In vitro luciferase reporter assays showed that PEX1 regulates the expression of MMP-9 by activating promoter. Furthermore, PEX1 gain- and loss-of-function experiments in rat neonatal cardiac fibroblasts and myocytes revealed that MMP-9 gene expression was affected by PEX1 predominantly in fibroblasts. Our results indicate that PEX1 is involved in regulating cardiac fibrosis and extracellular matrix turnover, particularly fibroblasts being responsible for the fibrosis-associated changes in gene expression. Furthermore, PEX1 activation of the MMP-9 promoter triggers the pro-fibrotic response directed by PEX1.

WDR12, a Member of Nucleolar PeBoW-Complex, Is Up-Regulated in Failing Hearts and Causes Deterioration of Cardiac Function.

AIMS: In a recent genome-wide association study, WD-repeat domain 12 (WDR12) was associated with early-onset myocardial infarction (MI). However, the function of WDR12 in the heart is unknown. METHODS AND RESULTS: We characterized cardiac expression of WDR12, used adenovirus-mediated WDR12 gene delivery to examine effects of WDR12 on left ventricular (LV) remodeling, and analyzed relationship between MI associated WDR12 allele and cardiac function in human subjects. LV WDR12 protein levels were increased in patients with dilated cardiomyopathy and rats post-infarction. In normal adult rat hearts, WDR12 gene delivery into the anterior wall of the LV decreased interventricular septum diastolic and systolic thickness and increased the diastolic and systolic diameters of the LV. Moreover, LV ejection fraction (9.1%, P<0.05) and fractional shortening (12.2%, P<0.05) were declined. The adverse effects of WDR12 gene delivery on cardiac function were associated with decreased cellular proliferation, activation of p38 mitogen-activated protein kinase (MAPK)/heat shock protein (HSP) 27 pathway, and increased protein levels of Block of proliferation 1 (BOP1), essential for ribosome biogenesis. Post-infarction WDR12 gene delivery decreased E/A ratio (32%, P<0.05) suggesting worsening of diastolic function. In human subjects, MI associated WDR12 allele was associated significantly with diastolic dysfunction and left atrial size. CONCLUSIONS: WDR12 triggers distinct deterioration of cardiac function in adult rat heart and the MI associated WDR12 variant is associated with diastolic dysfunction in human subjects.

Inhibition of Let-7 microRNA attenuates myocardial remodeling and improves cardiac function postinfarction in mice.

The members of lethal-7 (Let-7) microRNA (miRNA) family are involved in regulation of cell differentiation and reprogramming of somatic cells into induced pluripotent stem cells. However, their function in the heart is not known. In this study, we examined the effect of inhibiting the function of Let-7c miRNA on the progression of postinfarction left ventricular (LV) remodeling in mice. Myocardial infarction was induced with permanent ligation of left anterior descending coronary artery with a 4-week follow-up period. Let-7c miRNA was inhibited with a specific antagomir administered intravenously. The inhibition of Let-7c miRNA downregulated the levels of mature Let-7c miRNA and its other closely related members of Let-7 family in the heart and resulted in increased expression of pluripotency-associated genes Oct4 and Sox2 in cardiac fibroblasts in vitro and in adult mouse heart in vivo. Importantly, Let-7c inhibitor prevented the deterioration of cardiac function postinfarction, as demonstrated by preserved LV ejection fraction and elevated cardiac output. Improvement in cardiac function by Let-7c inhibitor postinfarction was associated with decreased apoptosis, reduced fibrosis, and reduction in the number of discoidin domain receptor 2-positive fibroblasts, while the number of c-kit(+) cardiac stem cells and Ki-67(+) proliferating cells remained unaltered. In conclusion, inhibition of Let-7 miRNA may be beneficial for the prevention of postinfarction LV remodeling and progression of heart failure.

Characterization of the regulatory mechanisms of activating transcription factor 3 by hypertrophic stimuli in rat cardiomyocytes.

AIMS: Activating transcription factor 3 (ATF3) is a stress-activated immediate early gene suggested to have both detrimental and cardioprotective role in the heart. Here we studied the mechanisms of ATF3 activation by hypertrophic stimuli and ATF3 downstream targets in rat cardiomyocytes. METHODS AND RESULTS: When neonatal rat cardiomyocytes were exposed to endothelin-1 (ET-1, 100 nM) and mechanical stretching in vitro, maximal increase in ATF3 expression occurred at 1 hour. Inhibition of extracellular signal-regulated kinase (ERK) by PD98059 decreased ET-1- and stretch-induced increase of ATF3 protein but not ATF3 mRNA levels, whereas protein kinase A (PKA) inhibitor H89 attenuated both ATF3 mRNA transcription and protein expression in response to ET-1 and stretch. To characterize further the regulatory mechanisms upstream of ATF3, p38 mitogen-activated protein kinase (MAPK) signaling was investigated using a gain-of-function approach. Adenoviral overexpression of p38α, but not p38ß, increased ATF3 mRNA and protein levels as well as DNA binding activity. To investigate the role of ATF3 in hypertrophic process, we overexpressed ATF3 by adenovirus-mediated gene transfer. In vitro, ATF3 gene delivery attenuated the mRNA transcription of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1), and enhanced nuclear factor-κB (NF-κB) and Nkx-2.5 DNA binding activities. Reduced PAI-1 expression was also detected in vivo in adult rat heart by direct intramyocardial adenovirus-mediated ATF3 gene delivery. CONCLUSIONS: These data demonstrate that ATF3 activation by ET-1 and mechanical stretch is partly mediated through ERK and cAMP-PKA pathways, whereas p38 MAPK pathway is involved in ATF3 activation exclusively through p38α isoform. ATF3 activation caused induction of modulators of the inflammatory response NF-κB and Nkx-2.5, as well as attenuation of pro-fibrotic and pro-inflammatory proteins IL-6 and PAI-1, suggesting cardioprotective role for ATF3 in the heart.

(Pro)renin receptor triggers distinct angiotensin II-independent extracellular matrix remodeling and deterioration of cardiac function.

BACKGROUND: Activation of the renin-angiotensin-system (RAS) plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (pro)renin receptor ((P)RR) is not yet solved. We determined here the direct functional and structural effects of (P)RR in the heart. METHODOLOGY/PRINCIPAL FINDINGS: (P)RR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (P)RR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01), fractional shortening (P<0.01), and intraventricular septum diastolic and systolic thickness, associated with approximately 2-fold increase in left ventricular (P)RR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (P)RR gene overexpression was mediated by angiotensin II (Ang II), we infused an AT(1) receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (P)RR overexpressing animals as well. Intramyocardial (P)RR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-ß1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (P)RR gene delivery was Ang II-dependent. Finally, (P)RR overexpression significantly increased direct protein-protein interaction between (P)RR and promyelocytic zinc-finger protein. CONCLUSIONS/SIGNIFICANCE: These results indicate for the first time that (P)RR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (P)RR as a novel therapeutic target to optimize RAS blockade in failing hearts.

Intramyocardial BNP gene delivery improves cardiac function through distinct context-dependent mechanisms.

BACKGROUND: B-type natriuretic peptide (BNP) is an endogenous peptide produced under physiological and pathological conditions mainly by ventricular myocytes. It has natriuretic, diuretic, blood pressure-lowering, and antifibrotic actions that could mediate cardiorenal protection in cardiovascular diseases. In the present study, we used BNP gene transfer to examine functional and structural effects of BNP on left ventricular (LV) remodeling. METHODS AND RESULTS: Human BNP was overexpressed by using adenovirus-mediated gene delivery in normal rat hearts and in hearts during the remodeling process after infarction and in an experimental model of angiotensin II-mediated hypertension. In healthy hearts, BNP gene delivery into the anterior wall of the LV decreased myocardial fibrosis (P<0.01, n=7 to 8) and increased capillary density (P<0.05, n=7 to 8) associated with a 7.3-fold increase in LV BNP peptide levels. Overexpression of BNP improved LV fractional shortening by 22% (P<0.05, n=6 to 7) and ejection fraction by 19% (P<0.05, n=6 to 7) after infarction. The favorable effect of BNP gene delivery on cardiac function after infarction was associated with normalization of cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression and phospholamban Thr17-phosphorylation. BNP gene delivery also improved fractional shortening and ejection fraction in angiotensin II-mediated hypertension as well as decreased myocardial fibrosis and LV collagen III mRNA levels but had no effect on angiogenesis or Ca(2+)-ATPase expression and phospholamban phosphorylation. CONCLUSIONS: Local intramyocardial BNP gene delivery improves cardiac function and attenuates adverse postinfarction and angiotensin II-induced remodeling. These results also indicate that myocardial BNP has pleiotropic, context-dependent, favorable actions on cardiac function and suggest that BNP acts locally as a key mechanical load-activated regulator of angiogenesis and fibrosis.

Characterization of molecular forms of N-terminal B-type natriuretic peptide in vitro.

BACKGROUND: The heterogeneity of circulating peptides may influence the interpretation of results from N-terminal profragment of BNP (NT-proBNP) assays. Our objective was to characterize the heterogeneity for better usability of the assays. METHODS: Endogenous proBNP was purified from patient samples and treated with trifluoromethanesulfonic acid (chemical deglycosylation). The human proBNP gene was introduced into rat hearts by adenoviral transfer. Cell lysates and plasma samples containing proBNP-derived peptides were analyzed by chromatography. The fate of exogenous recombinant NT-proBNP added to fresh whole blood samples was followed by immunoassays and chromatography. The main NT-proBNP components were isolated and identified by mass spectrometry. RESULTS: Immunoreactive NT-proBNP in human plasma comprised several molecular forms, as did circulating immunoreactive human NT-proBNP after adenoviral transfer of human proBNP cDNA into rat ventricular myocardium. Incubation of recombinant NT-proBNP(1-76) in human plasma or serum resulted in multiple components with the 2 major components identified as NT-proBNP(1-36) and NT-proBNP(1-62/64). Profiling by different antisera and chromatography indicated masking of the non-mid-region epitopes likely due to formation of oligomers. More than 75% of the original immunoreactivity in the mid-region epitope was retained after 3-week storage of plasma samples at room temperature. CONCLUSIONS: There is marked heterogeneity in immunoreactive NT-proBNP in plasma not related to glycosylation. The mid-region epitope of NT-proBNP is stable even in harsh storage conditions. Careful choice of antibody epitopes can yield extraordinarily robust assays.

Sorbitol dehydrogenase expression is regulated by androgens in the human prostate.

Sorbitol is an intermediate in the polyol pathway, which converts from glucose to fructose by sorbitol dehydrogenase (SORD). Androgens are essential for the development of prostate cancer. We studied castration-induced gene expression changes in the human prostate using the GeneChip array, and identified SORD as being androgen-regulated in the human prostate. A putative androgen-responsive regulatory region at the SORD 5' promoter was identified using promoter deletion constructs in a luciferase reporter assay in COS-7 cells. Chromatin immunoprecipitation assay was used to assess the binding of androgen receptor to suggested androgen responsive regulatory region. Finally, the expression of SORD in the human prostate was evaluated in 29 prostate tissue samples by immunohistochemistry. The expression of SORD decreased after castration. Androgen supplementation to the LNCaP prostate cancer cell line led to a 7.5-fold increase in SORD mRNA expression. Furthermore, a chromatin immunoprecipitation assay proved that the androgen receptor can bind to this putative androgen-responsive regulatory region. Finally, the expression of SORD in the human prostate was localised to epithelial cells of both benign and malignant prostate tissue by immunohistochemistry. In prostate cancer, increased immunostaining was associated with high Gleason patterns and high serum prostate-specific antigen concentrations. These results show that SORD is a novel androgen-regulated gene in the human prostate and suggest the need for more detailed analysis of the physiological role of SORD in the prostate.

Characterization of androgen-regulated expression of CYP3A5 in human prostate.

Testosterone is needed for the growth and development of the prostate. Androgen deprivation therapy is used for the treatment of prostate cancer. CYP3A5 is a human drug-metabolizing cytochrome P450 enzyme that metabolizes testosterone to the inactive 6beta-hydroxylated metabolite. We identified CYP3A5 as a novel androgen-regulated gene in human prostate by GeneChip analysis of human prostate tissues obtained from patients 3 days after therapeutic castration and from control patients. We further showed androgen induction of CYP3A5 messenger RNA (mRNA) in LNCaP prostate cancer cell line. Immunoblotting studies revealed CYP3A5 protein expression in all prostate samples studied. Immunohistochemistry and in situ hybridization was used for localization of CYP3A5 expression in prostate tissue. CYP3A5 was detected both in luminal and in basal epithelial cells of human prostate. Androgen response element was identified in the CYP3A5 proximal promoter and in electrophoretic mobility shift assay androgen receptor was found to bind this element. Androgen induction was abolished by mutation of the response element. We suggest that CYP3A5 is a part of an autoregulatory feedback loop controlling prostate cell exposure to androgens.