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1.
Arterioscler Thromb Vasc Biol ; 43(7): 1199-1218, 2023 07.
Article in English | MEDLINE | ID: mdl-37199159

ABSTRACT

BACKGROUND: Endothelial cells (ECs) are sensitive to physical forces created by blood flow, especially to laminar shear stress. Among the cell responses to laminar flow, EC polarization against the flow direction emerges as a key event, particularly during the development and remodeling of the vascular network. EC adopt an elongated planar cell shape with an asymmetrical distribution of intracellular organelles along the axis of blood flow. This study aimed to investigate the involvement of planar cell polarity via the receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) in endothelial responses to laminar shear stress. METHODS: We generated a genetic mouse model with EC-specific deletion of Ror2, in combination with in vitro approaches involving loss- and gain-of-function experiments. RESULTS: During the first 2 weeks of life, the endothelium of the mouse aorta undergoes a rapid remodeling associated with a loss of EC polarization against the flow direction. Notably, we found a correlation between ROR2 expression and endothelial polarization levels. Our findings demonstrate that deletion of Ror2 in murine ECs impaired their polarization during the postnatal development of the aorta. In vitro experiments further validated the essential role of ROR2 in both EC collective polarization and directed migration under laminar flow conditions. Exposure to laminar shear stress triggered the relocalization of ROR2 to cell-cell junctions where it formed a complex with VE-Cadherin and ß-catenin, thereby regulating adherens junctions remodeling at the rear and front poles of ECs. Finally, we showed that adherens junctions remodeling and cell polarity induced by ROR2 were dependent on the activation of the small GTPase Cdc42. CONCLUSIONS: This study identified ROR2/planar cell polarity pathway as a new mechanism controlling and coordinating collective polarity patterns of EC during shear stress response.


Subject(s)
Endothelial Cells , Receptor Tyrosine Kinase-like Orphan Receptors , Mice , Animals , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Cell Polarity/physiology , Endothelium, Vascular/metabolism , Intercellular Junctions , Stress, Mechanical
2.
Arterioscler Thromb Vasc Biol ; 42(5): 597-609, 2022 05.
Article in English | MEDLINE | ID: mdl-35387477

ABSTRACT

BACKGROUND: Genome-wide association studies have revealed robust associations of common genetic polymorphisms in an intron of the PHACTR-1 (phosphatase and actin regulator 1) gene (chr6p24), with cervical artery dissection, spontaneous coronary artery dissection, and fibromuscular dysplasia. The aim was to assess its role in the pathogenesis of cervical artery dissection or fibromuscular dysplasia. METHODS: Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells using 2 tissue-specific Cre-driver (PDGFB [platelet-derived growth factor B]-CreERT2 mice and Tie2 [tyrosine kinase with immunoglobulin and EGF homology domains]-Cre) and smooth muscle cells (smooth muscle actin-CreERT2) with a third tissue-specific Cre-driver. RESULTS: To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed first, a decrease in Phactr1 transcription and Phactr1 expression in endothelial cell and smooth muscle cell isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology, or actin organization were not different in knockout mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there were no more consequences of Phactr1 deletion during embryogenesis in endothelial cells. CONCLUSIONS: Loss of PHACTR-1 function in the cells involved in vascular physiology does not appear to induce a pathological vascular phenotype. The in vivo effect of the intronic variation described in genome-wide association studies is unlikely to involve downregulation in PHACTR-1 expression.


Subject(s)
Actins , Arterial Occlusive Diseases/metabolism , Fibromuscular Dysplasia , Microfilament Proteins/metabolism , Actins/metabolism , Animals , Endothelial Cells/metabolism , Fibromuscular Dysplasia/genetics , Genome-Wide Association Study , Mice , Mice, Knockout , Mice, Transgenic , Microfilament Proteins/genetics , Myocytes, Smooth Muscle/metabolism , Phosphoric Monoester Hydrolases/metabolism
3.
Arterioscler Thromb Vasc Biol ; 42(6): 745-763, 2022 06.
Article in English | MEDLINE | ID: mdl-35510550

ABSTRACT

BACKGROUND: While endothelial dysfunction is suggested to contribute to heart failure with preserved ejection fraction pathophysiology, understanding the importance of the endothelium alone, in the pathogenesis of diastolic abnormalities has not yet been fully elucidated. Here, we investigated the consequences of specific endothelial dysfunction on cardiac function, independently of any comorbidity or risk factor (diabetes or obesity) and their potential effect on cardiomyocyte. METHODS: The ubiquitine ligase Pdzrn3, expressed in endothelial cells (ECs), was shown to destabilize tight junction. A genetic mouse model in which Pdzrn3 is overexpressed in EC (iEC-Pdzrn3) in adults was developed. RESULTS: EC-specific Pdzrn3 expression increased cardiac leakage of IgG and fibrinogen blood-born molecules. The induced edema demonstrated features of diastolic dysfunction, with increased end-diastolic pressure, alteration of dP/dt min, increased natriuretic peptides, in addition to limited exercise capacity, without major signs of cardiac fibrosis and inflammation. Electron microscopic images showed edema with disrupted EC-cardiomyocyte interactions. RNA sequencing analysis of gene expression in cardiac EC demonstrated a decrease in genes coding for endothelial extracellular matrix proteins, which could be related to the fragile blood vessel phenotype. Irregularly shaped capillaries with hemorrhages were found in heart sections of iEC-Pdzrn3 mice. We also found that a high-fat diet was not sufficient to provoke diastolic dysfunction; high-fat diet aggravated cardiac inflammation, associated with an altered cardiac metabolic signature in EC-Pdzrn3 mice, reminiscent of heart failure with preserved ejection fraction features. CONCLUSIONS: An increase of endothelial permeability is responsible for mediating diastolic dysfunction pathophysiology and for aggravating detrimental effects of a high-fat diet on cardiac inflammation and metabolism.


Subject(s)
Cardiomyopathies , Heart Failure , Animals , Capillary Permeability , Endothelial Cells/metabolism , Fibrosis , Heart Failure/genetics , Heart Failure/metabolism , Inflammation/metabolism , Mice , Myocytes, Cardiac/metabolism , Stroke Volume/physiology , Ubiquitin-Protein Ligases
4.
Brain ; 145(6): 1992-2007, 2022 06 30.
Article in English | MEDLINE | ID: mdl-35511193

ABSTRACT

Cerebral small vessel disease is a leading cause of stroke and a major contributor to cognitive decline and dementia, but our understanding of specific genes underlying the cause of sporadic cerebral small vessel disease is limited. We report a genome-wide association study and a whole-exome association study on a composite extreme phenotype of cerebral small vessel disease derived from its most common MRI features: white matter hyperintensities and lacunes. Seventeen population-based cohorts of older persons with MRI measurements and genome-wide genotyping (n = 41 326), whole-exome sequencing (n = 15 965), or exome chip (n = 5249) data contributed 13 776 and 7079 extreme small vessel disease samples for the genome-wide association study and whole-exome association study, respectively. The genome-wide association study identified significant association of common variants in 11 loci with extreme small vessel disease, of which the chr12q24.11 locus was not previously reported to be associated with any MRI marker of cerebral small vessel disease. The whole-exome association study identified significant associations of extreme small vessel disease with common variants in the 5' UTR region of EFEMP1 (chr2p16.1) and one probably damaging common missense variant in TRIM47 (chr17q25.1). Mendelian randomization supports the causal association of extensive small vessel disease severity with increased risk of stroke and Alzheimer's disease. Combined evidence from summary-based Mendelian randomization studies and profiling of human loss-of-function allele carriers showed an inverse relation between TRIM47 expression in the brain and blood vessels and extensive small vessel disease severity. We observed significant enrichment of Trim47 in isolated brain vessel preparations compared to total brain fraction in mice, in line with the literature showing Trim47 enrichment in brain endothelial cells at single cell level. Functional evaluation of TRIM47 by small interfering RNAs-mediated knockdown in human brain endothelial cells showed increased endothelial permeability, an important hallmark of cerebral small vessel disease pathology. Overall, our comprehensive gene-mapping study and preliminary functional evaluation suggests a putative role of TRIM47 in the pathophysiology of cerebral small vessel disease, making it an important candidate for extensive in vivo explorations and future translational work.


Subject(s)
Brain Ischemia , Cerebral Small Vessel Diseases , Stroke , Animals , Brain Ischemia/complications , Cerebral Small Vessel Diseases/complications , Cerebral Small Vessel Diseases/diagnostic imaging , Cerebral Small Vessel Diseases/genetics , Endothelial Cells/pathology , Genome-Wide Association Study , Mice , Stroke/complications
5.
FASEB J ; 34(1): 1288-1303, 2020 01.
Article in English | MEDLINE | ID: mdl-31914666

ABSTRACT

Retinopathies remain major causes of visual impairment in diabetic patients and premature infants. Introduction of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) has transformed therapy for these proliferative retinopathies. However, limitations associated with anti-VEGF medications require to unravel new pathways of vessel growth to identify potential drug targets. Here, we investigated the role of Wnt/Frizzled-7 (Fzd7) pathway in a mouse model of oxygen-induced retinopathy (OIR). Using transgenic mice, which enabled endothelium-specific and time-specific Fzd7 deletion, we demonstrated that Fzd7 controls both vaso-obliteration and neovascular phases (NV). Deletion of Fzd7 at P12, after the ischemic phase of OIR, prevented formation of aberrant neovessels into the vitreous by suppressing proliferation of endothelial cells (EC) in tufts. Next we validated in vitro two Frd7 blocking strategies: a monoclonal antibody (mAbFzd7) against Fzd7 and a soluble Fzd7 receptor (CRD). In vivo a single intravitreal microinjection of mAbFzd7 or CRD significantly attenuated retinal neovascularization (NV) in mice with OIR. Molecular analysis revealed that Fzd7 may act through the activation of Wnt/ß-catenin and Jagged1 expression to control EC proliferation in extra-retinal neovessels. We identified Fzd7/ß-catenin signaling as new regulator of pathological retinal NV. Fzd7 appears to be a potent pharmacological target to prevent or treat aberrant angiogenesis of ischemic retinopathies.


Subject(s)
Diabetic Retinopathy/metabolism , Ischemia/metabolism , Repressor Proteins/metabolism , Retinal Neovascularization/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Diabetic Retinopathy/genetics , Diabetic Retinopathy/pathology , Gene Deletion , Ischemia/genetics , Ischemia/pathology , Jagged-1 Protein/biosynthesis , Jagged-1 Protein/genetics , Mice , Mice, Mutant Strains , Repressor Proteins/genetics , Retinal Neovascularization/genetics , Retinal Neovascularization/pathology , beta Catenin/genetics
6.
Haematologica ; 104(1): 70-81, 2019 01.
Article in English | MEDLINE | ID: mdl-30171023

ABSTRACT

Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.


Subject(s)
Endothelial Cells/metabolism , Janus Kinase 2/biosynthesis , P-Selectin/biosynthesis , Thrombosis/metabolism , Animals , Disease Models, Animal , Endothelial Cells/pathology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Hydroxyurea/pharmacology , Janus Kinase 2/genetics , Mice , Mice, Transgenic , P-Selectin/genetics , Thrombosis/drug therapy , Thrombosis/genetics , Thrombosis/pathology
7.
Biochim Biophys Acta Mol Cell Res ; 1864(7): 1142-1152, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28322931

ABSTRACT

The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the ß-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.


Subject(s)
RNA-Binding Proteins/metabolism , Wnt Signaling Pathway , Alternative Splicing , Animals , Cells, Cultured , HEK293 Cells , Humans , Male , Mice , Muscle, Skeletal/metabolism , Myocardium/metabolism , Protein Domains , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-Dawley , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolism
8.
Arterioscler Thromb Vasc Biol ; 36(12): 2369-2380, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27758766

ABSTRACT

OBJECTIVE: Vessel formation requires precise orchestration of a series of morphometric and molecular events controlled by a multitude of angiogenic factors and morphogens. Wnt/frizzled signaling is required for proper vascular formation. In this study, we investigated the role of the Fzd7 (frizzled-7) receptor in retinal vascular development and its relationship with the Wnt/ß-catenin canonical pathway and Notch signaling. APPROACH AND RESULTS: Using transgenic mice, we demonstrated that Fzd7 is required for postnatal vascular formation. Endothelial cell (EC) deletion of fzd7 (fzd7ECKO) delayed retinal plexus formation because of an impairment in tip cell phenotype and a decrease in stalk cell proliferation. Dvl (dishevelled) proteins are a main component of Wnt signaling and play a functionally redundant role. We found that Dvl3 depletion in dvl1-/- mice mimicked the fzd7ECKO vascular phenotype and demonstrated that Fzd7 acted via ß-catenin activation by showing that LiCl treatment rescued impairment in tip and stalk cell phenotypes induced in fzd7 mutants. Deletion of fzd7 or Dvl1/3 induced a strong decrease in Wnt canonical genes and Notch partners' expression. Genetic and pharmacological rescue strategies demonstrated that Fzd7 acted via ß-catenin activation, upstream of Notch signaling to control Dll4 and Jagged1 EC expression. CONCLUSIONS: Fzd7 expressed by EC drives postnatal angiogenesis via activation of Dvl/ß-catenin signaling and can control the integrative interaction of Wnt and Notch signaling during postnatal angiogenesis.


Subject(s)
Endothelial Cells/metabolism , Neovascularization, Physiologic , Receptors, G-Protein-Coupled/metabolism , Retinal Neovascularization/metabolism , Retinal Vessels/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Adaptor Proteins, Signal Transducing , Animals , Animals, Newborn , Calcium-Binding Proteins , Cell Proliferation , Cells, Cultured , Dishevelled Proteins/deficiency , Dishevelled Proteins/genetics , Endothelial Cells/drug effects , Frizzled Receptors , Genotype , Intracellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein/metabolism , Lithium Chloride/pharmacology , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic/drug effects , Phenotype , RNA Interference , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Receptors, Notch/metabolism , Retinal Neovascularization/genetics , Retinal Neovascularization/physiopathology , Retinal Vessels/drug effects , Transfection , Wnt Signaling Pathway/drug effects
9.
Circ Res ; 112(5): 762-70, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23343527

ABSTRACT

RATIONALE: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves. OBJECTIVE: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia. METHODS AND RESULTS: We induced hindlimb ischemia in wild-type and Dhh(-/-) mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh(-/-). By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle. CONCLUSIONS: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.


Subject(s)
Hedgehog Proteins/physiology , Hindlimb/blood supply , Ischemia/physiopathology , Neovascularization, Physiologic/physiology , Peripheral Nerves/physiology , Angiopoietin-1/metabolism , Animals , Cell Survival/physiology , Disease Models, Animal , Hedgehog Proteins/deficiency , Hedgehog Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Denervation , Muscle, Skeletal/innervation , Nerve Growth Factors/metabolism , Peripheral Nerves/cytology , Schwann Cells/cytology , Schwann Cells/physiology , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism
10.
Circ Res ; 110(1): 47-58, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22076635

ABSTRACT

RATIONALE: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive. OBJECTIVE: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development. METHODS AND RESULTS: We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and α-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network. CONCLUSIONS: These results suggest that Fzd4 is a major receptor involved in arterial formation and organization through a Wnt/PCP pathway.


Subject(s)
Arteries/cytology , Cell Polarity/physiology , Cell Proliferation , Frizzled Receptors/physiology , Neovascularization, Physiologic/physiology , Signal Transduction/physiology , Wnt Proteins/physiology , Adaptor Proteins, Signal Transducing/physiology , Animals , Arteries/physiology , Arterioles/cytology , Arterioles/physiology , Cell Movement/physiology , Dishevelled Proteins , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Frizzled Receptors/genetics , Gene Expression Regulation, Developmental/physiology , Gene Knock-In Techniques , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Microtubules/physiology , Models, Animal , Phosphoproteins/physiology
11.
Arterioscler Thromb Vasc Biol ; 31(11): e80-7, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21836067

ABSTRACT

OBJECTIVE: The inflammatory response after myocardial infarction plays a crucial role in the healing process. Lately, there is accumulating evidence that the Wnt/Frizzled pathway may play a distinct role in inflammation. We have shown that secreted frizzled-related protein-1 (sFRP-1) overexpression reduced postinfarction scar size, and we noticed a decrease in neutrophil infiltration in the ischemic tissue. We aimed to further elucidate the role of sFRP-1 in the postischemic inflammatory process. METHODS AND RESULTS: We found that in vitro, sFRP-1 was able to block leukocyte activation and cytokine production. We transplanted bone marrow cells (BMCs) from transgenic mice overexpressing sFRP-1 into wild-type recipient mice and compared myocardial healing with that of mice transplanted with wild-type BMCs. These results were compared with those obtained in transgenic mice overexpressing sFRP-1 specifically in endothelial cells or in cardiomyocytes to better understand the spatiotemporal mechanism of the sFRP-1 effect. Our findings indicate that when overexpressed in the BMCs, but not in endothelial cells or cardiomyocytes, sFRP-1 was able to reduce neutrophil infiltration after ischemia, by switching the balance of pro- and antiinflammatory cytokine expression, leading to a reduction in scar formation and better cardiac hemodynamic parameters. CONCLUSION: sFRP-1 impaired the loop of cytokine amplification and decreased neutrophil activation and recruitment into the scar, without altering the neutrophil properties. These data support the notion that sFRP-1 may be a novel antiinflammatory factor protecting the heart from damage after myocardial infarction.


Subject(s)
Cicatrix/etiology , Cicatrix/metabolism , Inflammation/metabolism , Myocardial Infarction/complications , Myocardial Infarction/metabolism , Proteins/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Bone Marrow Transplantation , Cell Line , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Cicatrix/pathology , Cytokines/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Mice , Mice, Transgenic , Models, Animal , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Neutrophils/drug effects , Neutrophils/pathology , Proteins/pharmacology
12.
Front Physiol ; 13: 906272, 2022.
Article in English | MEDLINE | ID: mdl-35874523

ABSTRACT

Heart failure with preserved ejection fraction (HFpEF) has been recognized as the greatest single unmet need in cardiovascular medicine. Indeed, the morbi-mortality of HFpEF is high and as the population ages and the comorbidities increase, so considerably does the prevalence of HFpEF. However, HFpEF pathophysiology is still poorly understood and therapeutic targets are missing. An unifying, but untested, theory of the pathophysiology of HFpEF, proposed in 2013, suggests that cardiovascular risk factors lead to a systemic inflammation, which triggers endothelial cells (EC) and coronary microvascular dysfunction. This cardiac small vessel disease is proposed to be responsible for cardiac wall stiffening and diastolic dysfunction. This paradigm is based on the fact that microvascular dysfunction is highly prevalent in HFpEF patients. More specifically, HFpEF patients have been shown to have decreased cardiac microvascular density, systemic endothelial dysfunction and a lower mean coronary flow reserve. Importantly, impaired coronary microvascular function has been associated with the severity of HF. This review discusses evidence supporting the causal role of endothelial dysfunction in the pathophysiology of HFpEF in human and experimental models.

13.
Article in English | MEDLINE | ID: mdl-35074794

ABSTRACT

The Wnt/frizzled signaling pathway is one of the major regulators of endothelial biology, controlling key cellular activities. Many secreted Wnt ligands have been identified and can initiate diverse signaling via binding to a complex set of Frizzled (Fzd) transmembrane receptors and coreceptors. Roughly, Wnt signaling is subdivided into two pathways: the canonical Wnt/ß-catenin signaling pathway whose main downstream effector is the transcriptional coactivator ß-catenin, and the noncanonical Wnt signaling pathway, which is subdivided into the Wnt/Ca2+ pathway and the planar cell polarity pathway. Here, we will focus on its cross talk with other angiogenic pathways and on its role in blood-retinal- and blood-brain-barrier formation and its maintenance in a differentiated state. We will unravel how retinal vascular pathologies and neurovascular degenerative diseases result from disruption of the Wnt pathway related to vascular instability, and highlight current research into therapeutic options.


Subject(s)
Blood-Brain Barrier , Wnt Signaling Pathway , Endothelium , Frizzled Receptors , Humans , Ligands
14.
J Cereb Blood Flow Metab ; 42(4): 613-629, 2022 04.
Article in English | MEDLINE | ID: mdl-34644209

ABSTRACT

Blood brain barrier (BBB) disruption is a critical component of the pathophysiology of cognitive impairment of vascular etiology (VCI) and associated with Alzheimer's disease (AD). The Wnt pathway plays a crucial role in BBB maintenance, but there is limited data on its role in cognitive pathologies. The E3 ubiquitin ligase PDZRN3 is a regulator of the Wnt pathway. In a murine model of VCI, overexpressing Pdzrn3 in endothelial cell (EC) exacerbated BBB hyperpermeability and accelerated cognitive decline. We extended these observations, in both VCI and AD models, showing that EC-specific depletion of Pdzrn3, reinforced the BBB, with a decrease in vascular permeability and a subsequent spare in cognitive decline. We found that in cerebral vessels, Pdzrn3 depletion protects against AD-induced Wnt target gene alterations and enhances endothelial tight junctional proteins. Our results provide evidence that Wnt signaling could be a molecular link regulating BBB integrity and cognitive decline under VCI and AD pathologies.


Subject(s)
Alzheimer Disease , Blood-Brain Barrier , Ubiquitin-Protein Ligases , Alzheimer Disease/metabolism , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Capillary Permeability , Endothelial Cells/metabolism , Homeostasis , Mice , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
15.
Sci Rep ; 12(1): 8, 2022 01 07.
Article in English | MEDLINE | ID: mdl-34996942

ABSTRACT

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes (CM) connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between CM. Yet no factor on its own is known to coordinate CM polarized organization. We have previously shown that PDZRN3, an ubiquitine ligase E3 expressed in various tissues including the heart, mediates a branch of the Planar cell polarity (PCP) signaling involved in tissue patterning, instructing cell polarity and cell polar organization within a tissue. PDZRN3 is expressed in the embryonic mouse heart then its expression dropped significantly postnatally corresponding with heart maturation and CM polarized elongation. A moderate CM overexpression of Pdzrn3 (Pdzrn3 OE) during the first week of life, induced a severe eccentric hypertrophic phenotype with heart failure. In models of pressure-overload stress heart failure, CM-specific Pdzrn3 knockout showed complete protection against degradation of heart function. We reported that Pdzrn3 signaling induced PKC ζ expression, c-Jun nuclear translocation and a reduced nuclear ß catenin level, consistent markers of the planar non-canonical Wnt signaling in CM. We then show that subcellular localization (intercalated disk) of junction proteins as Cx43, ZO1 and Desmoglein 2 was altered in Pdzrn3 OE mice, which provides a molecular explanation for impaired CM polarization in these mice. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of CM, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.


Subject(s)
Heart Failure/enzymology , Heart Failure/prevention & control , Heart/growth & development , Ubiquitin-Protein Ligases/metabolism , Animals , Heart Failure/genetics , Heart Failure/physiopathology , Humans , Male , Mice , Mice, Knockout , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/genetics , beta Catenin/genetics , beta Catenin/metabolism
16.
Circ Res ; 104(9): 1095-102, 2009 May 08.
Article in English | MEDLINE | ID: mdl-19342601

ABSTRACT

We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.


Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetic Foot/surgery , Fetal Stem Cells/transplantation , Ischemia/complications , Lower Extremity/blood supply , Neovascularization, Physiologic , Stem Cell Transplantation , Wnt Proteins/metabolism , Wound Healing , AC133 Antigen , Animals , Antigens, CD/analysis , Aorta/embryology , Cell Differentiation , Cell Movement , Cell Proliferation , Cell Survival , Cells, Cultured , Culture Media, Conditioned/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Experimental/surgery , Diabetic Foot/etiology , Diabetic Foot/metabolism , Diabetic Foot/physiopathology , Fetal Stem Cells/immunology , Fetal Stem Cells/metabolism , Glycoproteins/analysis , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-8/metabolism , Ischemia/metabolism , Ischemia/physiopathology , Ischemia/surgery , Male , Membrane Proteins/metabolism , Mice , Paracrine Communication , Peptides/analysis , Signal Transduction , Time Factors , Vascular Endothelial Growth Factor A/metabolism
17.
Mol Ther ; 18(8): 1545-52, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20551912

ABSTRACT

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.


Subject(s)
Hindlimb/metabolism , Hindlimb/pathology , Ischemia/therapy , Mesenchymal Stem Cell Transplantation/methods , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Wnt Proteins/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Movement/genetics , Cell Movement/physiology , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Ischemia/metabolism , Ischemia/pathology , Mice , Mice, Knockout , Myoblasts/cytology , Myoblasts/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Wnt Proteins/genetics , Wnt4 Protein
18.
Arterioscler Thromb Vasc Biol ; 29(12): 2090-2, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19745199

ABSTRACT

OBJECTIVE: Studying the mechanisms of neovascularization and evaluating the effects of proangiogenic strategies require accurate analysis of the neovascular network. We sought to evaluate the contribution of the microcomputed tomography (mCT) providing high-resolution 3-dimensional (3D) structural data, to a better comprehension of the well-studied mouse hindlimb postischemic neovascularization. METHODS AND RESULTS: We showed a predominant arteriogenesis process in the thigh and a predominant angiogenesis-related process in the tibiofibular region, in response to ischemia during the first 15 days. After 15 days, mCT quantitative analysis reveals a remodeling of arterial neovessels and a regression depending on the restoration of the blood flow. We provided also new mCT data on the rapid and potent angiogenic effects of mesenchymal stem cell therapy on vessel formation and organization. We discussed the contribution of this technique compared with or in addition to data generated by the more conventional approaches. CONCLUSIONS: This study demonstrated that optimized mCT is a robust method for providing new insights into the 3D understanding of postischemic vessel formation.


Subject(s)
Hindlimb/blood supply , Ischemia/pathology , Neovascularization, Physiologic , Tomography, X-Ray Computed/methods , Animals , Barium , Contrast Media , Disease Models, Animal , Imaging, Three-Dimensional , Ischemia/surgery , Mesenchymal Stem Cell Transplantation , Mice , Neoprene , Peripheral Vascular Diseases/pathology , Peripheral Vascular Diseases/surgery
19.
Stem Cells ; 26(11): 2991-3001, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18757297

ABSTRACT

Mesenchymal stem cell (MSC) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/beta-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled-related protein-1 (sFRP1), a modulator of the Wnt/Fz pathway, could modify MSC capacities, endowing MSC to increase vessel maturation. In the engraftment model, we show that murine bone marrow-derived MSC induced a beneficial vascular effect through a direct cellular contribution to vascular cells. MSC quickly organized into primitive immature vessel tubes connected to host circulation; this organization preceded host endothelial cell (EC) and smooth muscle cell (SMC) recruitment to later form mature neovessel. MSC sustained neovessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neovessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased platelet-derived growth factor-BB (PDGF-BB) expression in MSC and enhanced beta-catenin-dependent cell-cell contacts between MSC themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neovessels and vessel maturation through a glycogen synthase kinase 3 beta (GSK3beta)-dependent pathway. sFRP1-overexpressing MSC compared with control MSC were well elongated and in a closer contact with the vascular wall, conditions required to achieve an organized mature vessel wall. We propose that genetically modifying MSC to overexpress sFRP1 may be potentially effective in promoting therapeutic angiogenesis/arteriogenesis processes. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Mesenchymal Stem Cells/cytology , Neovascularization, Physiologic/physiology , Proteins/metabolism , Animals , Becaplermin , Cattle , Cell Adhesion/physiology , Cells, Cultured , Collagen , Drug Combinations , Endothelial Cells/cytology , Endothelial Cells/physiology , Intracellular Signaling Peptides and Proteins , Laminin , Lentivirus/genetics , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , Mice, Nude , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , Platelet-Derived Growth Factor/metabolism , Proteins/genetics , Proteoglycans , Proto-Oncogene Proteins c-sis , Transplantation, Heterologous , beta Catenin/metabolism
20.
Arterioscler Thromb Vasc Biol ; 28(12): 2131-6, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18772499

ABSTRACT

OBJECTIVE: Estradiol (E(2)) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERalpha(-/-) mice have previously shown that E(2) acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E(2) to mediate endothelial repair are still poorly understood. METHODS AND RESULTS: We show here that after endovascular carotid artery injury, E(2)-enhanced endothelial repair is lost in osteopontin-deficient mice (OPN(-/-)). Transplantation of OPN(-/-) bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E(2). In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E(2)-enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E(2) enhances bone marrow-derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin. CONCLUSIONS: We demonstrate here that E(2) acceleration of the endothelial repair requires osteopontin, both for bone marrow-derived cell recruitment and for endothelial cell migration and proliferation.


Subject(s)
Carotid Artery Injuries/physiopathology , Endothelial Cells/cytology , Endothelial Cells/physiology , Estradiol/pharmacology , Osteopontin/physiology , Animals , Bone Marrow Transplantation , Carotid Artery Injuries/drug therapy , Carotid Artery Injuries/pathology , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Female , Mice , Mice, Knockout , Mice, Transgenic , Osteopontin/deficiency , Osteopontin/genetics , Regeneration/drug effects , Regeneration/physiology
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