Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis.

Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.

A quantitative 3D intravital look at the juxtaglomerular renin-cell-niche reveals an individual intra/extraglomerular feedback system.

The juxtaglomerular niche occupied by renin cells (RCN) plays an important role in glomerular repair but the precise temporal and spatial interrelations remain unclear. This study proposes the hypothesis of a local intra-extraglomerular regenerative feedback system and establishes a new quantifiable system for RCN responses in individual glomeruli in vivo. A strictly intraglomerular two-photon laser-induced injury model was established. Labeled renin cells (RC) in transgenic renin reporter mice were fate-traced in healthy and injured glomeruli over several days by intravital microscopy and quantified via new three-dimensional image processing algorithms based on ray tracing. RC in healthy glomeruli demonstrated dynamic extraglomerular protrusions. Upon intraglomerular injury the corresponding RCN first increased in volume and then increased in area of dynamic migration up to threefold compared to their RCN. RC started migration reaching the site of injury within 3 hours and acquired a mesangial cell phenotype without losing physical RCN-contact. During intraglomerular repair only the corresponding RCN responded via stimulated neogenesis, a process of de novo differentiation of RC to replenish the RCN. Repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular RCN repair feedback system in individual glomeruli in vivo in a quantifiable manner.

Intravital imaging of hemodynamic glomerular effects of enalapril or/and empagliflozin in STZ-diabetic mice.

Background: Diabetic kidney disease is the leading cause of end-stage renal disease. Administration of ACE inhibitors or/and SGLT2 inhibitors show renoprotective effects in diabetic and other kidney diseases. The underlying renoprotective mechanisms of SGLT2 inhibition, especially in combination with ACE inhibition, are incompletely understood. We used longitudinal intravital microscopy to directly elucidate glomerular hemodynamics on a single nephron level in response to the ACE inhibitor enalapril or/and the SGLT2 inhibitor empagliflozin. Methods: Five weeks after the induction of diabetes by streptozotocin, male C57BL/6 mice were treated with enalapril, empagliflozin, enalapril/empagliflozin or placebo for 3 days. To identify hemodynamic regulation mechanisms, longitudinal intravital multiphoton microscopy was employed to measure single nephron glomerular filtration rate (snGFR) and afferent/efferent arteriole width. Results: Diabetic mice presented a significant hyperfiltration. Compared to placebo treatment, snGFR was reduced in response to enalapril, empagliflozin, or enalapril/empagliflozin administration under diabetic conditions. While enalapril treatment caused significant dilation of the efferent arteriole (12.55 ± 1.46 µm vs. control 11.92 ± 1.04 µm, p < 0.05), empagliflozin led to a decreased afferent arteriole diameter (11.19 ± 2.55 µm vs. control 12.35 ± 1.32 µm, p < 0.05) in diabetic mice. Unexpectedly under diabetic conditions, the combined treatment with enalapril/empagliflozin had no effects on both afferent and efferent arteriole diameter change. Conclusion: SGLT2 inhibition, besides ACE inhibition, is an essential hemodynamic regulator of glomerular filtration during diabetes mellitus. Nevertheless, additional mechanisms-independent from hemodynamic regulation-are involved in the nephroprotective effects especially of the combination therapy and should be further explored in future studies.

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion.

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.