Publications

In epithelial and muscle cells, intermediate filaments (IFs) are important for resistance to mechanical stress. The aim of this study was to elucidate whether IFs are also important for providing resistance to mechanical stress in the Müller cells of the retina and whether this has any pathophysiological consequences. We used mice deficient in IF proteins glial fibrillary acidic protein and/or vimentin (GFAP–/–, Vim–/– and GFAP–/–Vim–/–), and stress on the retina was applied by excision of the eyes immediately post mortem (compared with in situ fixation) or by inducing a neovascular response to oxygen-induced retinopathy (OIR). The structure of unchallenged retinas was normal, but mechanical stress caused local separation of the inner limiting membrane (ILM) and adjacent tissue from the rest of the retina in GFAP–/–Vim–/– mice and, to a lesser extent, in Vim–/– mice. This detachment occurred within the endfeet of Müller cells, structures normally rich in IFs but IF-free in GFAP–/–Vim–/– mice. Hypoxia-induced neovascularization was comparable in all groups of mice with respect to the retinal surface area occupied by new vessels. However, the vessels traversed the ILM and penetrated the vitreous body less frequently than in wild-type retinas (31-55% in Vim–/–, 66-79% in GFAP–/–Vim–/–). We conclude that IFs are important for maintaining the mechanical integrity of Müller-cell endfeet and the inner retinal layers under a mechanical challenge. Furthermore, the absence of IFs in Müller cells leads to an abnormal response of the vascular system to ischemia, specifically decreased ability of newly formed blood vessels to traverse the ILM.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH) 2 vitamin D [1,25(OH) 2 D 3 ] action on calcium homeostasis. We assessed the rate of serum 45 Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D 9K expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA 1b ) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH) 2 D 3 injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D 9K expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D 9K and PMCA 1b expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D 9K , should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B 186 , is a neuroprotective factor. VEGF-B 186 protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B 186 were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B 186 prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B 186 was safer and did not cause vessel growth or blood–brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.

Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

Significance Statement The specialized vessels comprising the renal vasculature are characterized by highly differentiated renal endothelial cell types, but this heterogeneity has been poorly inventoried. Using single-cell RNA sequencing, the authors developed a high-resolution atlas of mouse renal endothelial cells. They also investigated how medullary renal endothelial cells adapt to a switch from diuresis to antidiuresis. This study describes the molecular and metabolic adaptation of medullary renal endothelial cells to dehydration, and uncovers a role for mitochondrial oxidative phosphorylation in hyperosmolarity conditions to allow for urine concentration. The authors’ atlas of mouse renal endothelial cells provides a resource for future studies, and their findings may provide insights into cardiometabolic or kidney diseases involving hyperosmolarity and dehydration, in which urine concentration capacity is perturbed. Background Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. Methods We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo . Results We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. Conclusions This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

<p>Supplementary Figure 7 - Effects of VEGFR-1 signaling ablation on IGF-1R/IR signaling pathways in PAN02 and E0771 tumors implanted in obese mice; Supplementary Figure 8 - Additional effects of VEGFR-1 signaling ablation and metformin on tumor metabolism and vessel density in obese mice.</p>