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.

Angiopoietin-like 4-Induced 3D Capillary Morphogenesis Correlates to Stabilization of Endothelial Adherens Junctions and Restriction of VEGF-Induced Sprouting.

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell-cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.

Scavenger Receptor Cysteine-Rich domains of Lysyl Oxidase-Like2 regulate endothelial ECM and angiogenesis through non-catalytic scaffolding mechanisms.

Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic cross-linking activity.

VEGF-A plasma levels are associated with microvascular obstruction in patients with ST-segment elevation myocardial infarction.

BACKGROUND: Microvascular obstruction (MVO) is associated with poor outcome after ST-segment elevation myocardial infarction (STEMI). Vascular endothelial growth factor-A (VEGF-A) is a vascular permeability inducer playing a key role in MVO pathogenesis. We aimed to assess whether VEGF-A levels are associated with MVO, when evaluated by magnetic resonance imaging (MRI) in STEMI patients. METHODS: The multicenter prospective PREGICA study included a CMR substudy with all consecutive patients with a first STEMI who had undergone cardiac MRI at baseline and at 6-month follow-up. Patients with initial TIMI flow >1 were excluded. VEGF-A levels were measured in blood samples drawn at inclusion. RESULTS: Between 2010 and 2017, 147 patients (mean age 57 ±â€¯10 years; 84% males) were included. MVO was present in 65 (44%) patients. After multivariate analysis, higher troponin peak (OR 1.005; 95% CI 1.001-1.008; p = 0.007) and VEGF-A levels (OR 1.003; 95% CI 1.001-1.005; p = 0.015) were independently associated with MVO. When considering only patients with successful percutaneous coronary intervention (final TIMI flow 3, n = 130), higher troponin peak (p = 0.004) and VEGF-A levels (p = 0.03) remained independently predictive of MVO. Moreover, MVO was associated with adverse left ventricular (LV) remodeling and VEGF-A levels were significantly and inversely correlated with LV ejection fraction (EF) at 6-month follow-up. CONCLUSION: Our results show that VEGF-A levels were independently associated with MVO during STEMI and correlated with mid-term LVEF alteration. VEGF-A could therefore be considered as a biomarker of MVO in STEMI patients and be used to stratify patient prognosis.

The interaction of heparan sulfate proteoglycans with endothelial transglutaminase-2 limits VEGF165-induced angiogenesis.

Sprouting angiogenesis is stimulated by vascular endothelial growth factor (VEGF165) that is localized in the extracellular matrix (ECM) and binds to heparan sulfate (HS)-bearing proteins known as heparan sulfate proteoglycans (HSPGs). VEGF165 presentation by HSPGs enhances VEGF receptor-2 (VEGFR2) signaling. We investigated the effect of TG2, which binds to HSPGs, on the interaction between VEGF165 and HS and angiogenesis. Mice with tg2 deficiency showed transiently enhanced retina vessel formation and increased vascularization of VEGF165-containing Matrigel implants. In addition, endothelial cells in which TG2 was knocked down exhibited enhanced VEGF165-induced sprouting and migration, which was associated with increased phosphorylation of VEGFR2 at Tyr(951) and its targets Src and Akt. TG2 knockdown did not affect the phosphorylation of VEGFR2 at Tyr(1175) or cell proliferation in response to VEGF165 and sprouting or signaling in response to VEGF121. Decreased phosphorylation of VEGFR2 at Tyr(951) was due to ECM-localized TG2, which reduced the binding of VEGF165 to endothelial ECM in a manner that required its ability to bind to HS but not its catalytic activity. Surface plasmon resonance assays demonstrated that TG2 impeded the interaction between VEGF165 and HS. These results show that TG2 controls the formation of VEGF165-HSPG complexes and suggest that this regulation could be pharmacologically targeted to modulate developmental and therapeutic angiogenesis.

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.

Interaction of the coiled-coil domain with glycosaminoglycans protects angiopoietin-like 4 from proteolysis and regulates its antiangiogenic activity.

Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.