Preventing VEGF-Mediated Vascular Permeability by Experimentally Potentiating BBB Characteristics in Endothelial Cells.

Difficulties with poor reproducibility and translatability of animal model-based research, along with increased efforts to abide by the 3Rs tenet of animal welfare, are driving demand for more relevant human cellular systems. This is especially true for central nervous system (CNS) vasculatures with specialized properties and barriers, namely the blood-brain and blood-retinal barriers (BBB and BRB, respectively) which are difficult to model in vitro. The BBB and BRB protect neurovascular units by regulating nutrient homeostasis, maintaining local ion levels, protecting against exposure from circulating toxins and pathogens, and restricting passage of peripheral immune factors. In this manuscript, we will describe transgenic and pharmacological-based protocols to generate relevant BBB and BRB models both from human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and from primary human umbilical vein endothelial cells (HUVECs). When followed, researchers can expect to generate well-characterized, anatomical and functional BBB and BRB EC monolayers in 36-48 h that are stable up to 90 h. The ability to generate more relevant BBB and BRB EC cultures will improve drug discovery efforts and inform future therapies for neurovascular disorders.

Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age-related macular degeneration.

Choroidal neovascularization (CNV) is a major cause of visual impairment in patients suffering from wet age-related macular degeneration (AMD), particularly when refractory to intraocular anti-VEGF injections. Here we report that treatment with the oral mineralocorticoid receptor (MR) antagonist spironolactone reduces signs of CNV in patients refractory to anti-VEGF treatment. In animal models of wet AMD, pharmacological inhibition of the MR pathway or endothelial-specific deletion of MR inhibits CNV through VEGF-independent mechanisms, in part through upregulation of the extracellular matrix protein decorin. Intravitreal injections of spironolactone-loaded microspheres and systemic delivery lead to similar reductions in CNV. Together, our work suggests MR inhibition as a novel therapeutic option for wet AMD patients unresponsive to anti-VEGF drugs.

Adult nephron-specific MR-deficient mice develop a severe renal PHA-1 phenotype.

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR(Pax8/LC1)) using a doxycycline-inducible system. Under standard diet, MR(Pax8/LC1) mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.