Publications

Endothelial cells (ECs) are more than inert blood vessel lining material. Instead, they are active players in the formation of new blood vessels (angiogenesis) both in health and (life-threatening) diseases. Recently, a new concept arose by which EC metabolism drives angiogenesis in parallel to well-established angiogenic growth factors (e.g., vascular endothelial growth factor). 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3-driven glycolysis generates energy to sustain competitive behavior of the ECs at the tip of a growing vessel sprout, whereas carnitine palmitoyltransferase 1a-controlled fatty acid oxidation regulates nucleotide synthesis and proliferation of ECs in the stalk of the sprout. To maintain vascular homeostasis, ECs rely on an intricate metabolic wiring characterized by intracellular compartmentalization, use metabolites for epigenetic regulation of EC subtype differentiation, crosstalk through metabolite release with other cell types, and exhibit EC subtype-specific metabolic traits. Importantly, maladaptation of EC metabolism contributes to vascular disorders, through EC dysfunction or excess angiogenesis, and presents new opportunities for anti-angiogenic strategies. Here we provide a comprehensive overview of established as well as newly uncovered aspects of EC metabolism.

Background —Chronic hypoxia results in the development of pulmonary hypertension and subsequent right heart failure. A role of the plasminogen system in the pathogenesis of pulmonary hypertension and pulmonary vascular remodeling has been suggested. Methods and Results —Mice with targeted deficiency of the gene encoding tissue-type plasminogen activator (t-PA −/− ), urokinase-type plasminogen activator (u-PA −/− ), u-PA receptor (u-PAR −/− ), or plasminogen (plg −/− ) were subjected to hypoxic conditions. Hypoxia caused a significant 2.5-fold rise in right ventricular pressure in wild-type mice. Deficiency of u-PA or plasminogen prevented this increase in right ventricular pressure, t-PA −/− mice showed changes that were fully comparable with wild-type mice, and u-PAR −/− mice showed a partial response. Hypoxia induced an increase in smooth muscle cells within pulmonary arterial walls and a vascular rarefaction in the lungs of wild-type but not of u-PA −/− or plg −/− mice. Elastic lamina fragmentation, observed in hypoxic wild-type but not in u-PA or plasminogen-deficient mice, suggested that proliferation of vascular smooth muscle cells was dependent on u-PA–mediated elastic membrane degradation. Hypoxia-induced right ventricular remodeling in wild-type mice, characterized by cardiomyocyte hypertrophy and increased collagen contents, was not seen in u-PA −/− and plg −/− mice. Conclusions —Loss of the u-PA or plasminogen gene protects against the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling. These observations point to an essential role of u-PA–mediated plasmin generation in the adaptive response to chronic hypoxia and the occurrence of hypoxic pulmonary vascular disease.

Abstract In the pathogenesis of sepsis and disseminated intravascular coagulation (DIC), dysfunctional anticoagulant pathways are important. The function of the protein C system in DIC is impaired because of low levels of protein C and down-regulation of thrombomodulin. The administration of (activated) protein C results in an improved outcome in experimental and clinical studies of DIC. It is unknown whether congenital deficiencies in the protein C system are associated with more severe DIC. The aim of the present study was to investigate the effect of a heterozygous deficiency of protein C on experimental DIC in mice. Mice with single-allele targeted disruption of the protein C gene (PC+/–) mice and wild-type littermates (PC+/+) were injected with Escherichia coli endotoxin (50 mg/kg) intraperitoneally. PC+/–mice had more severe DIC, as evidenced by a greater decrease in fibrinogen level and a larger drop in platelet count. Histologic examination showed more fibrin deposition in lungs, kidneys, and liver in mice with a heterozygous deficiency of protein C. Interestingly, PC+/– mice had significantly higher levels of proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, indicating an interaction between the protein C system and the inflammatory response. Survival was lower at 12 and 24 hours after endotoxin in the PC+/– mice. These results confirm the important role of the protein C system in the coagulative-inflammatory response on endotoxemia and may suggest that congenital deficiencies in the protein C system are associated with more severe DIC and adverse outcome in sepsis.

Results are displayed as fold induction of the phosphorylated/total p38 ratio in SEMA3B cells versus controls, as measured by densitometric analysis. (B) Serum-starved MDA-MB435 cells were treated for 30 min with CM collected from SEMA3B or EV control cells. The levels of phospho-p38 and phospho–ATF-2 were analyzed in protein lysates by immunoblotting. The same filter was furthermore decorated to reveal vinculin, providing loading controls. Plotted values indicate fold increase of the phosphorylated p38/vinculin or ATF2/vinculin ratios in SEMA3B-treated cells versus controls. (C) Immunoblotting analysis of A549 cells treated as in B with SEMA3B or EV control CM. Filters were probed to reveal phospho-p38, phospho-p42/44–MAPK, and vinculin levels (as loading control). The graph shows fold increase of the phosphorylated p38/vinculin or phospho-p42/44–MAPK/vinculin ratios in SEMA3B-treated cells versus controls. (D) MAB208 antibody was used to detect IL-8 expression by immunofluorescence (top) or by Western blotting (bottom) in SEMA3B-expressing and control cells grown in the absence of serum for 48 h, in the presence or absence of 10 μM of the selective p38–MAPK inhibitor SB202190. Micrographs were acquired with a bioimager (Pathway; BD Biosciences) and quantified by Autovision 1.5 (10 images per each data point). Results are displayed as mean ± SD. ***, P &lt; 0.001. (E) CDKI p21 expression was detected by Western blotting in A549 cells, transduced with SEMA3B or EV control, upon serum starvation (48 h in 1% FCS). Cells treated with 20 nM staurosporin (STS) for 24 h provided a positive control for p21 activation. β-Actin levels provided a reference for protein loading. Plotted values indicate fold induction of the p21/β-actin ratio. (F) A549 cells were serum starved for 24 h and then treated for 30 min with CM from SEMA3B or EV control cells in the presence of 20 ng/ml anti-NP1 blocking antibody or the unrelated antibody anti–VSV-G. Cells treated with 20 μm cisplatin for 2 h provided a positive control for p38 activation. The levels of phospho-p38 and vinculin (as loading control) were analyzed in protein lysates by immunoblotting. (G) Consistent with a previous report, many tumor cells could not survive RNAi-mediated knock down of NP1 (reference ); however, we managed to stably express NP1-targeted shRNAs (shNP1) in HeLa cells previously transduced with SEMA3B or EV (). Real-time PCR analysis indicated that NP1 expression levels were knocked down to 20% compared with cells transduced with unrelated sequences (shCTR; not depicted). Notably, SEMA3B autocrine stimulation was unable to up-regulate IL-8 levels in NP1-deficient cells, indicating the requirement for this receptor in the signaling pathway. The graph shows fold increase of expression relative to control cells. Data shown are the mean ± SEM.<b>Copyright information:</b>Taken from "The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment"The Journal of Experimental Medicine 2008;205(5):1155-1171.Published online 12 May 2008PMCID:PMC2373847.

Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism.

<b>Rationale:</b> Congenital diaphragmatic hernia (CDH) results in lung hypoplasia. In severe cases, tracheal occlusion (TO) can be offered to promote lung growth. However, the benefit is limited, and novel treatments are required to supplement TO. VEGF (vascular endothelial growth factor) is downregulated in animal models of CDH and could be a therapeutic target, but its role in human CDH is not known. <b>Objectives:</b> To investigate whether VEGF supplementation could be a suitable treatment for CDH-associated lung pathology. <b>Methods:</b> Fetal lungs from patients with CDH were used to determine pulmonary morphology and VEGF expression. A novel human <i>ex vivo</i> model of fetal lung compression recapitulating CDH features was developed and used to determine the effect of exogenous VEGF supplementation. A nanoparticle-based approach for intrapulmonary delivery of VEGF was developed by conjugating it on functionalized nanodiamonds, which was tested in experimental CDH <i>in vivo</i>. <b>Measurements and Main Results:</b> VEGF expression was downregulated in the distal pulmonary epithelium of human CDH fetuses in conjunction with attenuated cell proliferation. The compression model resulted in impaired branching morphogenesis similar to CDH and downregulation of VEGF expression in conjunction with reduced proliferation of terminal bud epithelial progenitors; these could be reversed by exogenous supplementation of VEGF. Prenatal delivery of VEGF with the functionalized nanodiamond VEGF platform in CDH fetal rats resulted in lung growth and pulmonary arterial remodeling that was complementary to that achieved by TO alone with appearances comparable to healthy controls. <b>Conclusions:</b> This innovative approach could have a significant impact on the treatment of CDH.

Significance Statement In previous work, the authors demonstrated that short-term loss of integrity of the luminal part of the glycocalyx envelope that covers endothelial cells resulted in impaired glomerular filtration barrier function. In this study, using knockout mice lacking endothelial hyaluronan synthase 2 (the enzyme that produces hyaluronan, the main structural component of the glycocalyx layer), they found that loss of glomerular endothelial hyaluronan leads to mesangiolysis and glomerulosclerosis. Tissue from patients with diabetic nephropathy showed loss of glomerular endothelial hyaluronan in association with lesion formation. The authors also demonstrated that hyaluronan loss results in disturbed signaling of the extracellular matrix stabilizer angiopoietin 1. These findings suggest that the loss of glomerular endothelial hyaluronan in diabetic nephropathy may be a new therapeutic target to restabilize glomerular lesions. Background A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. Methods To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. Results Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. Conclusions Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca 2+ influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca 2+ influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

Abstract Background There are no validated biomarkers to guide patient selection for treatment with the anti-VEGF antibody bevacizumab. We recently identified a locus, consisting of 4 SNPs in the VEGFR-1 TK domain, that correlated with progression-free (PFS) and overall survival (OS) of bevacizumab-treated pancreatic cancer patients in the AViTA phase III trial. The causal SNP underlying this association and its effect on VEGFR-1 expression or function are not yet known. Methods We conducted a fine-mapping study using data from the 1000 Genomes Project to identify the causal variant in this locus. The candidate SNP was functionally validated by studying effects on VEGFR-1 RNA (RT-PCR) or protein (ELISA) expression. Transcription and translation were measured using transient transfection and rabbit reticulocyte assays. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy newborns. Association of causal SNPs was replicated in bevacizumab-treated renal cancer patients from the AVOREN phase III trial via Sequenom genotyping. Results Fine-mapping identified 48 common SNPs linked to one of the 4 SNPs in the VEGFR-1 locus. In silico functional prediction revealed that only one SNP (rs7993418) was located in exon 28. No other SNPs were located in exons or functionally important regions. Remarkably, rs7993418 is a synonymous SNP introducing a shift in codon usage (TAT to TAC codon) at an evolutionary conserved tyrosine (Tyr1213Tyr). In vitro transcription/translation of VEGFR-1 cDNAs carrying either the wildtype TAT or mutant TAC codon revealed that both constructs transcribe equal amounts of mRNA, whereas a 27% increased VEGFR-1 protein expression was found for TAC versus TAT-carrying constructs (P&amp;lt;0.001). Overexpression of both cDNAs in HEK293T cells demonstrated a similar increase in VEGFR-1 protein, but not mRNA expression for the TAC construct (P&amp;lt;0.001). Since transmembrane and soluble VEGFR-1 levels strongly correlate, we studied the effect of rs7993418 on sVEGFR-1 expression in human plasma. We found 18% and 19% increased median sVEGFR-1 levels in homozygous carriers of the TAC codon in two independent cohorts (P=0.006 and P=0.014). Primary HUVECs homozygous for the TAC codon also showed increased tmVEGFR-1 and sVEGFR-1 protein expression (P=0.049 and P=0.044), leading to increased VEGFR-1 downstream ERK1/2 signaling upon stimulation with the VEGFR-1 specific ligand PlGF (P&amp;lt;0.05). Finally, association of rs7993418 with bevacizumab treatment outcome was also replicated in AVOREN (per-allele HR=1.8; P=0.033). Conclusions The synonymous rs7993418 SNP (Tyr1213Tyr) is a functional SNP that potentially underlies the association of the VEGFR-1 locus with bevacizumab treatment outcome. In particular, rs7993418 causes a shift in codon usage, leading to increased VEGFR-1 expression and enhanced downstream signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-447. doi:1538-7445.AM2012-LB-447

Abstract Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3−/−) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1−/−) or PHD2 (PHD2+/−). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α– and NF-κB–mediated enhancement of the innate immune response.

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.