Systemwide effects of ER-intracellular membrane contact site disturbance in primary endothelial cells.

Membrane contact sites (MCS) make up a crucial route of inter-organelle non-vesicular transport within the cell. Multiple proteins are involved in this process, which includes the ER-resident proteins vesicle associated membrane protein associated protein A and -B (VAPA/B) that form MCS between the ER and other membrane compartments. Currently most functional data on VAP depleted phenotypes have shown alterations in lipid homeostasis, induction of ER stress, dysfunction of UPR and autophagy, as well as neurodegeneration. Literature on concurrent silencing of VAPA/B is still sparse; therefore, we investigated how it affects the macromolecule pools of primary endothelial cells. Our transcriptomics results showed significant upregulation in genes related to inflammation, ER and Golgi dysfunction, ER stress, cell adhesion, as well as Coat Protein Complex-I and -II (COP-I, COP-II) vesicle transport. Genes related to cellular division were downregulated, as well as key genes of lipid and sterol biosynthesis. Lipidomics analyses revealed reductions in cholesteryl esters, very long chain highly unsaturated and saturated lipids, whereas increases in free cholesterol and relatively short chain unsaturated lipids were evident. Furthermore, the knockdown resulted in an inhibition of angiogenesis in vitro. We speculate that ER MCS depletion has led to multifaceted outcomes, which include elevated ER free cholesterol content and ER stress, alterations in lipid metabolism, ER-Golgi function and vesicle transport, which have led to a reduction in angiogenesis. The silencing also induced an inflammatory response, consistent with upregulation of markers of early atherogenesis. To conclude, ER MCS mediated by VAPA/B play a crucial role in maintaining cholesterol traffic and sustain normal endothelial functions.

Alterations of Cardiac Protein Kinases in Cyclic Nucleotide-Dependent Signaling Pathways in Human Ischemic Heart Failure.

Objectives: Impaired protein kinase signaling is a hallmark of ischemic heart disease (IHD). Inadequate understanding of the pathological mechanisms limits the development of therapeutic approaches. We aimed to identify the key cardiac kinases and signaling pathways in patients with IHD with an effort to discover potential therapeutic strategies. Methods: Cardiac kinase activity in IHD left ventricle (LV) and the related signaling pathways were investigated by kinomics, transcriptomics, proteomics, and integrated multi-omics approach. Results: Protein kinase A (PKA) and protein kinase G (PKG) ranked on top in the activity shift among the cardiac kinases. In the IHD LVs, PKA activity decreased markedly compared with that of controls (62% reduction, p = 0.0034), whereas PKG activity remained stable, although the amount of PKG protein increased remarkably (65%, p = 0.003). mRNA levels of adenylate cyclases (ADCY 1, 3, 5, 9) and cAMP-hydrolysing phosphodiesterases (PDE4A, PDE4D) decreased significantly, although no statistically significant alterations were observed in that of PKGs (PRKG1 and PRKG2) and guanylate cyclases (GUCYs). The gene expression of natriuretic peptide CNP decreased remarkably, whereas those of BNP, ANP, and neprilysin increased significantly in the IHD LVs. Proteomics analysis revealed a significant reduction in protein levels of "Energy metabolism" and "Muscle contraction" in the patients. Multi-omics integration highlighted intracellular signaling by second messengers as the top enriched Reactome pathway. Conclusion: The deficiency in cAMP/PKA signaling pathway is strongly implicated in the pathogenesis of IHD. Natriuretic peptide CNP could be a potential therapeutic target for the modulation of cGMP/PKG signaling.

Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes.

BACKGROUND: Thyroid hormone responsive protein (THRSP) is a lipogenic nuclear protein that is highly expressed in murine adipose tissue, but its role in humans remains unknown. METHODS: We characterized the insulin regulation of THRSP in vivo in human adipose tissue biopsies and in vitro in Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. To this end, we measured whole-body insulin sensitivity using the euglycemic insulin clamp technique in 36 subjects [age 40 ± 9 years, body mass index (BMI) 27.3 ± 5.0 kg/m2]. Adipose tissue biopsies were obtained at baseline and after 180 and 360 min of euglycemic hyperinsulinemia for measurement of THRSP mRNA concentrations. To identify functions affected by THRSP, we performed a transcriptomic analysis of THRSP-silenced SGBS adipocytes. Mitochondrial function was assessed by measuring mitochondrial respiration as well as oxidation and uptake of radiolabeled oleate and glucose. Lipid composition in THRSP silencing was studied by lipidomic analysis. RESULTS: We found insulin to increase THRSP mRNA expression 5- and 8-fold after 180 and 360 min of in vivo euglycemic hyperinsulinemia. This induction was impaired in insulin-resistant subjects, and THRSP expression was closely correlated with whole-body insulin sensitivity. In vitro, insulin increased both THRSP mRNA and protein concentrations in SGBS adipocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. A transcriptomic analysis of THRSP-silenced adipocytes showed alterations in mitochondrial functions and pathways of lipid metabolism, which were corroborated by significantly impaired mitochondrial respiration and fatty acid oxidation. A lipidomic analysis revealed decreased hexosylceramide concentrations, supported by the transcript concentrations of enzymes regulating sphingolipid metabolism. CONCLUSIONS: THRSP is regulated by insulin both in vivo in human adipose tissue and in vitro in adipocytes, and its expression is downregulated by insulin resistance. As THRSP silencing decreases mitochondrial respiration and fatty acid oxidation, its downregulation in human adipose tissue could contribute to mitochondrial dysfunction. Furthermore, disturbed sphingolipid metabolism could add to metabolic dysfunction in obese adipose tissue.

Global effects of pharmacologic inhibition of OSBP in human umbilical vein endothelial cells.

Oxysterol-binding protein (OSBP) is a cholesterol/PI4P exchanger at contacts of the endoplasmic reticulum (ER) with trans-Golgi network (TGN) and endosomes. Several central endothelial cell (EC) functions depend on adequate cholesterol distribution in cellular membranes. Here we elucidated the effects of pharmacologic OSBP inhibition on the lipidome and transcriptome of human umbilical vein endothelial cells (HUVECs). OSBP was inhibited for 24 h with 25 nM Schweinfurthin G (SWG) or Orsaponin (OSW-1), followed by analyses of cellular cholesterol, 27-hydroxy-cholesterol, and triacylglycerol concentration, phosphatidylserine synthesis rate, the lipidome, as well as lipid droplet staining and western analysis of OSBP protein. Next-generation RNA sequencing of the SWG-treated and control HUVECs and angiogenesis assays were performed. Protein-normalized lipidomes of the inhibitor-treated cells revealed decreases in glycerophospholipids, the most pronounced effect being on phosphatidylserines and the rate of their synthesis, as well as increases in cholesteryl esters, triacylglycerols and lipid droplet number. Transcriptome analysis of SWG-treated cells suggested ER stress responses apparently caused by disturbed cholesterol exit from the ER, as indicated by suppression of cholesterol biosynthetic genes. OSBP was associated with the TGN in the absence of inhibitors and disappeared therefrom in inhibitor-treated cells in a time-dependent manner, coinciding with OSBP reduction on western blots. Prolonged treatment with SWG or OSW-1 inhibited angiogenesis in vitro. To conclude, inhibition of OSBP in primary endothelial cells induced multiple effects on the lipidome, transcriptome changes suggesting ER stress, and disruption of in vitro angiogenic capacity. Thus, OSBP is a crucial regulator of EC lipid homeostasis and angiogenic capacity.

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis.

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.

Coordination of inter-organelle communication and lipid fluxes by OSBP-related proteins.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute one of the largest families of lipid-binding/transfer proteins (LTPs) in eukaryotes. The current view is that many of them mediate inter-organelle lipid transfer over membrane contact sites (MCS). The transfer occurs in several cases in a 'counter-current' fashion: A lipid such as cholesterol or phosphatidylserine (PS) is transferred against its concentration gradient driven by transport of a phosphoinositide in the opposite direction. In this way ORPs are envisioned to maintain the distinct organelle lipid compositions, with impacts on multiple organelle functions. However, the functions of ORPs extend beyond lipid homeostasis to regulation of processes such as cell survival, proliferation and migration. Important expanding areas of mammalian ORP research include their roles in viral and bacterial infections, cancers, and neuronal function. The yeast OSBP homologue (Osh) proteins execute multifaceted functions in sterol and glycerophospholipid homeostasis, post-Golgi vesicle transport, phosphatidylinositol-4-phosphate, sphingolipid and target of rapamycin (TOR) signalling, and cell cycle control. These observations identify ORPs as lipid transporters and coordinators of signals with an unforeseen variety of cellular processes. Understanding their activities not only enlightens the biology of the living cell but also allows their employment as targets of new therapeutic approaches for disease.

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells.

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.

ANGPTL3 deficiency alters the lipid profile and metabolism of cultured hepatocytes and human lipoproteins.

Loss-of-function (LOF) mutations in ANGPTL3, an inhibitor of lipoprotein lipase (LPL), cause a drastic reduction of serum lipoproteins and protect against the development of atherosclerotic cardiovascular disease. Therefore, ANGPTL3 is a promising therapy target. We characterized the impacts of ANGPTL3 depletion on the immortalized human hepatocyte (IHH) transcriptome, lipidome and human plasma lipoprotein lipidome. The transcriptome of ANGPTL3 knock-down (KD) cells showed altered expression of several pathways related to lipid metabolism. Accordingly, ANGPTL3 depleted IHH displayed changes in cellular overall fatty acid (FA) composition and in the lipid species composition of several lipid classes, characterized by abundant n-6 and n-3 polyunsaturated FAs (PUFAs). This PUFA increase coincided with an elevation of lipid mediators, among which there were species relevant for resolution of inflammation, protection from lipotoxic and hypoxia-induced ER stress, hepatic steatosis and insulin resistance or for the recovery from cardiovascular events. Cholesterol esters were markedly reduced in ANGPTL3 KD IHH, coinciding with suppression of the SOAT1 mRNA and protein. ANGPTL3 LOF caused alterations in plasma lipoprotein FA and lipid species composition. All lipoprotein fractions of the ANGPTL3 LOF subjects displayed a marked drop of 18:2n-6, while several highly unsaturated triacylglycerol (TAG) species were enriched. The present work reveals distinct impacts of ANGPTL3 depletion on the hepatocellular lipidome, transcriptome and lipid mediators, as well as on the lipidome of lipoproteins isolated from plasma of ANGPTL3-deficient human subjects. It is important to consider these lipidomics and transcriptomics findings when targeting ANGPTL3 for therapy and translating it to the human context.