Blood endothelium transition and phenotypic plasticity: A key regulator of integrity/permeability in response to ischemia.

In the human body, the 1013 blood endothelial cells (ECs) which cover a surface of 500-700 m2 (Mai et al., 2013) are key players of tissue homeostasis, remodeling and regeneration. Blood vessel ECs play a major role in the regulation of metabolic and gaz exchanges, cell trafficking, blood coagulation, vascular tone, blood flow and fluid extravasation (also referred to as blood vascular permeability). ECs are heterogeneous in various capillary beds and have the exquisite capacity to cope with environmental changes by regulating their gene expression. Ischemia has major detrimental effects on the endothelium and ischemia-induced regulation of vascular integrity is of paramount importance for human health, as small amounts of fluid accumulation in the interstitium may be responsible for major effects on organ functions and patients outcome. In this review, we will here focus on the stimuli and the molecular mechanisms that control blood endothelium maintenance and phenotypic plasticity/transition involved in controlling blood capillary leakage that might open new avenues for therapeutic applications.

Blood Monocyte Phenotype Is A Marker of Cardiovascular Risk in Type 2 Diabetes.

BACKGROUND: Diabetes is a major risk factor for atherosclerotic cardiovascular diseases with a 2-fold higher risk of cardiovascular events in people with diabetes compared with those without. Circulating monocytes are inflammatory effector cells involved in both type 2 diabetes (T2D) and atherogenesis. METHODS: We investigated the relationship between circulating monocytes and cardiovascular risk progression in people with T2D, using phenotypic, transcriptomic, and metabolomic analyses. cardiovascular risk progression was estimated with coronary artery calcium score in a cohort of 672 people with T2D. RESULTS: Coronary artery calcium score was positively correlated with blood monocyte count and frequency of the classical monocyte subtype. Unsupervised k-means clustering based on monocyte subtype profiles revealed 3 main endotypes of people with T2D at varying risk of cardiovascular events. These observations were confirmed in a validation cohort of 279 T2D participants. The predictive association between monocyte count and major adverse cardiovascular events was validated through an independent prospective cohort of 757 patients with T2D. Integration of monocyte transcriptome analyses and plasma metabolomes showed a disruption of mitochondrial pathways (tricarboxylic acid cycle, oxidative phosphorylation pathway) that underlined a proatherogenic phenotype. CONCLUSIONS: In this study, we provide evidence that frequency and monocyte phenotypic profile are closely linked to cardiovascular risk in patients with T2D. The assessment of monocyte frequency and count is a valuable predictive marker for risk of cardiovascular events in patients with T2D. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04353869.

Three-vessel coronary infusion of cardiosphere-derived cells for the treatment of heart failure with preserved ejection fraction in a pre-clinical pig model.

Heart failure with preserved ejection fraction (HFpEF) is a major public health concern. Its outcome is poor and, as of today, barely any treatments have been able to decrease its morbidity or mortality. Cardiosphere-derived cells (CDCs) are heart cell products with anti-fibrotic, anti-inflammatory and angiogenic properties. Here, we tested the efficacy of CDCs in improving left ventricular (LV) structure and function in pigs with HFpEF. Fourteen chronically instrumented pigs received continuous angiotensin II infusion for 5 weeks. LV function was investigated through hemodynamic measurements and echocardiography at baseline, after 3 weeks of angiotensin II infusion before three-vessel intra-coronary CDC (n = 6) or placebo (n = 8) administration and 2 weeks after treatment (i.e., at completion of the protocol). As expected, arterial pressure was significantly and similarly increased in both groups. This was accompanied by LV hypertrophy that was not affected by CDCs. LV systolic function remained similarly preserved during the whole protocol in both groups. In contrast, LV diastolic function was impaired (increases in Tau, LV end-diastolic pressure as well as E/A, E/E'septal and E/E'lateral ratios) but CDC treatment significantly improved all of these parameters. The beneficial effect of CDCs on LV diastolic function was not explained by reduced LV hypertrophy or increased arteriolar density; however, interstitial fibrosis was markedly reduced. Three-vessel intra-coronary administration of CDCs improves LV diastolic function and reduces LV fibrosis in this hypertensive model of HFpEF.

FX06 to rescue SARS-CoV-2-induced acute respiratory distress syndrome: a randomized clinical trial.

BACKGROUND: Vascular leakage is a major feature of acute respiratory distress syndrome (ARDS). We aimed to evaluate the efficacy of FX06, a drug under development that stabilizes interendothelial cell junctions, at reducing vascular leakage during SARS-CoV-2-induced ARDS. METHODS: This multicenter, double-blinded, randomized trial included adults with COVID-19-associated ARDS who had received invasive mechanical ventilation for < 5 days and were randomized to receive either intravenous FX06 (400 mg/d, for 5 days) or its vehicle as placebo. The primary endpoint was the lowering-from day 1 to day 7-of the transpulmonary thermodilution-derived extravascular lung-water index (EVLWi). RESULTS: Twenty-five patients were randomized to receive FX06 and 24 the placebo. Although EVLWi was elevated at baseline (median [IQR] 15.6 mL/kg [13.5; 18.5]), its declines from day 1 to day 7 were comparable for FX06 recipients and controls (respectively, - 1.9 [- 3.3; - 0.5] vs. - 0.8 [- 5.5; - 1.1] mL/kg; estimated effect - 0.8 [- 3.1; + 2.4], p = 0.51). Cardiac indexes, pulmonary vascular permeability indexes, and fluid balances were also comparable, as were PaO2/FiO2 ratios and durations of mechanical ventilation. Adverse event rates were similar for the 2 groups, although more FX06 recipients developed ventilator-associated pneumonia (16/25 (64%) vs. 6/24 (24%), p = 0.009). CONCLUSIONS: In this unique-dosing-regimen study, FX06 did not lower SARS-CoV-2-induced pulmonary vascular leakage. Future investigations will need to evaluate its efficacy at earlier times during the disease or using other regimens. Trial registration NCT04618042. Registered 5 November 2020.

Dynamics of Endothelial Engagement and Filopodia Formation in Complex 3D Microscaffolds.

The understanding of endothelium-extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars ("open"), but not "closed" hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.

Extracellular matrix scaffolding in angiogenesis and capillary homeostasis.

The extracellular matrix (ECM) of blood vessels, which is composed of both the vascular basement membrane (BM) and the interstitial ECM is identified as a crucial component of the vasculature. We here focus on the unique molecular composition and scaffolding of the capillary ECM, which provides structural support to blood vessels and regulates properties of endothelial cells and pericytes. The major components of the BM are collagen IV, laminins, heparan sulfate proteoglycans and nidogen and also associated proteins such as collagen XVIII and fibronectin. Their organization and scaffolding in the BM is required for proper capillary morphogenesis and maintenance of vascular homeostasis. The BM also regulates vascular mechanosensing. A better understanding of the mechanical and structural properties of the vascular BM and interstitial ECM therefore opens new perspectives to control physiological and pathological angiogenesis and vascular homeostasis. The overall aim of this review is to explain how ECM scaffolding influences angiogenesis and capillary integrity.

Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.-Schmelzer, C. E. H., Heinz, A., Troilo, H., Lockhart-Cairns, M.-P., Jowitt, T. A., Marchand, M. F., Bidault, L., Bignon, M., Hedtke, T., Barret, A., McConnell, J. C., Sherratt, M. J., Germain, S., Hulmes, D. J. S., Baldock, C., Muller, L. Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Experimental Model of Human Malignant Mesothelioma in Athymic Mice.

Malignant pleural mesothelioma (MPM) is a thoracic aggressive cancer caused by asbestos exposure, which is difficult to diagnose and treat. Here, we characterized an in vivo orthotopic xenograft model consisting of human mesothelioma cells (designed as H2052/484) derived from a pleural NCI-H2052 tumor injected in partially immunodeficient athymic mice. We assessed tumor formation and tumor-dependent patterns of inflammation. H2052/484 cells conserved their mesothelioma phenotype and most characteristics from the parental NCI-H2052 cells. After intra-thoracic injection of H2052/484 cells, thoracic tumors developed in nearly all mice (86%) within 14 days, faster than from parental NCI-H2052 cells. When the mice were euthanized, the pleural lavage fluid was examined for immune cell profiles. The pleural immune cell population increased with tumor development. Interestingly, the proportion of myeloid-derived suppressor cell and macrophage (especially CD206⁺ M2 macrophages) populations increased in the pleural fluid of mice with large mesothelioma development, as previously observed in immunocompetent mice. This reliable orthotopic model recapitulates human mesothelioma and may be used for the study of new treatment strategies.

ANGPTL4-αvß3 interaction counteracts hypoxia-induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enhanced angiogenesis and increased cardiac perfusion after myocardial infarction in protein tyrosine phosphatase 1B-deficient mice.

The protein tyrosine phosphatase 1B (PTP1B) modulates tyrosine kinase receptors, among which is the vascular endothelial growth factor receptor type 2 (VEGFR2), a key component of angiogenesis. Because PTP1B deficiency in mice improves left ventricular (LV) function 2 mo after myocardial infarction (MI), we hypothesized that enhanced angiogenesis early after MI via activated VEGFR2 contributes to this improvement. At 3 d after MI, capillary density was increased at the infarct border of PTP1B(-/-) mice [+7±2% vs. wild-type (WT), P = 0.05]. This was associated with increased extracellular signal-regulated kinase 2 phosphorylation and VEGFR2 activation (i.e., phosphorylated-Src/Src/VEGFR2 and dissociation of endothelial VEGFR2/VE-cadherin), together with higher infiltration of proangiogenic M2 macrophages within unchanged overall infiltration. In vitro, we showed that PTP1B inhibition or silencing using RNA interference increased VEGF-induced migration and proliferation of mouse heart microvascular endothelial cells as well as fibroblast growth factor (FGF)-induced proliferation of rat aortic smooth muscle cells. At 8 d after MI in PTP1B(-/-) mice, increased LV capillary density (+21±3% vs. WT; P<0.05) and an increased number of small diameter arteries (15-50 µm) were likely to participate in increased LV perfusion assessed by magnetic resonance imaging and improved LV compliance, indicating reduced diastolic dysfunction. In conclusion, PTP1B deficiency reduces MI-induced heart failure promptly after ischemia by enhancing angiogenesis, myocardial perfusion, and diastolic function.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke.

AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.

Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4.

BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

Connection between cardiac vascular permeability, myocardial edema, and inflammation during sepsis: role of the α1AMP-activated protein kinase isoform.

OBJECTIVE: As adenosine monophosphate (AMP)-activated protein kinase both controls cytoskeleton organization in endothelial cells and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall edema during sepsis. DESIGN: Controlled animal study. SETTINGS: University research laboratory. SUBJECTS: C57BL/6J, α1AMPK, and α1AMPK mice. INTERVENTION: Sepsis was triggered in vivo using a sublethal injection of lipopolysaccharide (O55B5, 10 mg/kg), inducing systolic left ventricular dysfunction. Left ventricular function, edema, vascular permeability, and inflammation were assessed in vivo in both wild-type mice (α1AMPK) and α1AMP-activated protein kinase-deficient mice (α1AMPK). The 5-aminoimidazole-4-carboxamide riboside served to study the impact of AMP-activated protein kinase activation on vascular permeability in vivo. The integrity of endothelial cell monolayers was also examined in vitro after lipopolysaccharide challenge in the presence of aminoimidazole-4-carboxamide riboside and/or after α1AMP-activated protein kinase silencing. MEASUREMENTS AND MAIN RESULTS: α1AMP-activated protein kinase deficiency dramatically impaired tolerance to lipopolysaccharide challenge. Indeed, α1AMPK exhibited heightened cardiac vascular permeability after lipopolysaccharide challenge compared with α1AMPK. Consequently, an increase in left ventricular mass corresponding to exaggerated wall edema occurred in α1AMPK, without any further decrease in systolic function. Mechanistically, the lipopolysaccharide-induced α1AMPK cardiac phenotype could not be attributed to major changes in the systemic inflammatory response but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMP-activated protein kinase activation by aminoimidazole-4-carboxamide riboside counteracted lipopolysaccharide-induced hyperpermeability in wild-type mice in vivo as well as in endothelial cells in vitro. This effect was associated with a potent protection of zonula occludens-1 linear border pattern in endothelial cells. CONCLUSIONS: Our results demonstrate for the first time the involvement of a signaling pathway in the control of left ventricular wall edema during sepsis. AMP-activated protein kinase exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated left ventricular wall edema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in patients with sepsis.

Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Implantation of engineered human microvasculature to study human infectious diseases in mouse models.

Animal models for studying human pathogens are crucially lacking. We describe the implantation in mice of engineered human mature microvasculature consisting of endothelial and perivascular cells embedded in collagen hydrogel that allows investigation of pathogen interactions with the endothelium, including in vivo functional studies. Using Neisseria meningitidis as a paradigm of human-restricted infection, we demonstrated the strength and opportunities associated with the use of this approach.

Alteration of developmental and pathological retinal angiogenesis in angptl4-deficient mice.

Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.

Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration.

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.