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1.
Circulation ; 142(17): 1647-1662, 2020 10 27.
Article in English | MEDLINE | ID: mdl-32820662

ABSTRACT

BACKGROUND: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. METHODS: We utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia. RESULTS: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP+ CD144+ CD11b- fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP+ CD11b- cells from the plug revealed a small subset of YFP+ CD144+ CD11b- E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. CONCLUSIONS: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.


Subject(s)
Fibroblasts/pathology , Hindlimb/blood supply , Ischemia/pathology , Animals , Disease Models, Animal , Humans , Male , Mice , Neovascularization, Physiologic
2.
Circulation ; 138(9): 913-928, 2018 08 28.
Article in English | MEDLINE | ID: mdl-29545372

ABSTRACT

BACKGROUND: Angiogenesis is integral for embryogenesis, and targeting angiogenesis improves the outcome of many pathological conditions in patients. TBX20 is a crucial transcription factor for embryonic development, and its deficiency is associated with congenital heart disease. However, the role of TBX20 in angiogenesis has not been described. METHODS: Loss- and gain-of-function approaches were used to explore the role of TBX20 in angiogenesis both in vitro and in vivo. Angiogenesis gene array was used to identify key downstream targets of TBX20. RESULTS: Unbiased gene array survey showed that TBX20 knockdown profoundly reduced angiogenesis-associated PROK2 (prokineticin 2) gene expression. Indeed, loss of TBX20 hindered endothelial cell migration and in vitro angiogenesis. In a murine angiogenesis model using subcutaneously implanted Matrigel plugs, we observed that TBX20 deficiency markedly reduced PROK2 expression and restricted intraplug angiogenesis. Furthermore, recombinant PROK2 administration enhanced angiogenesis and blood flow recovery in murine hind-limb ischemia. In zebrafish, transient knockdown of tbx20 by morpholino antisense oligos or genetic disruption of tbx20 by CRISPR/Cas9 impaired angiogenesis. Furthermore, loss of prok2 or its cognate receptor prokr1a also limited angiogenesis. In contrast, overexpression of prok2 or prokr1a rescued the impaired angiogenesis in tbx20-deficient animals. CONCLUSIONS: Our study identifies TBX20 as a novel transcription factor regulating angiogenesis through the PROK2-PROKR1 (prokineticin receptor 1) pathway in both development and disease and reveals a novel mode of angiogenic regulation whereby the TBX20-PROK2-PROKR1 signaling cascade may act as a "biological capacitor" to relay and sustain the proangiogenic effect of vascular endothelial growth factor. This pathway may be a therapeutic target in the treatment of diseases with dysregulated angiogenesis.


Subject(s)
Gastrointestinal Hormones/metabolism , Ischemia/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Neuropeptides/metabolism , Receptors, G-Protein-Coupled/metabolism , T-Box Domain Proteins/metabolism , Zebrafish Proteins/metabolism , Angiogenesis Inducing Agents/pharmacology , Animals , Animals, Genetically Modified , Disease Models, Animal , Gastrointestinal Hormones/genetics , Gene Expression Regulation, Developmental , Hindlimb , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Ischemia/drug therapy , Ischemia/genetics , Ischemia/physiopathology , Male , Mice, Inbred NOD , Mice, SCID , Neovascularization, Physiologic/drug effects , Neuropeptides/genetics , Receptors, G-Protein-Coupled/genetics , Recombinant Proteins/pharmacology , Signal Transduction , T-Box Domain Proteins/genetics , Zebrafish , Zebrafish Proteins/genetics
3.
J Med Chem ; 53(24): 8650-62, 2010 Dec 23.
Article in English | MEDLINE | ID: mdl-21090716

ABSTRACT

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.


Subject(s)
Aldehyde-Lyases/antagonists & inhibitors , Antirheumatic Agents/chemical synthesis , Imidazoles/chemical synthesis , Isoxazoles/chemical synthesis , Oximes/chemical synthesis , Aldehyde-Lyases/genetics , Animals , Antirheumatic Agents/pharmacokinetics , Antirheumatic Agents/pharmacology , Arthritis, Experimental/drug therapy , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Blood Pressure/drug effects , Cell Movement , Dogs , Heart Rate/drug effects , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , Isoxazoles/pharmacokinetics , Isoxazoles/pharmacology , Lymphocytes/drug effects , Lymphocytes/physiology , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Oximes/pharmacokinetics , Oximes/pharmacology , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship
4.
J Immunol ; 176(3): 1899-907, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16424221

ABSTRACT

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1-/- mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1-/- macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.


Subject(s)
Cell Cycle Proteins/physiology , Down-Regulation/physiology , Immediate-Early Proteins/physiology , Immunity, Innate/physiology , Phosphoprotein Phosphatases/physiology , Protein Tyrosine Phosphatases/physiology , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Bone Marrow Cells/immunology , Cell Cycle Proteins/genetics , Cells, Cultured , Dual Specificity Phosphatase 1 , Extracellular Signal-Regulated MAP Kinases/physiology , Immediate-Early Proteins/deficiency , Immediate-Early Proteins/genetics , Incidence , Macrophages/immunology , Mice , Mice, Knockout , Phenotype , Phosphoprotein Phosphatases/deficiency , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 1 , Protein Tyrosine Phosphatases/deficiency , Protein Tyrosine Phosphatases/genetics , Severity of Illness Index , Toll-Like Receptors/physiology , p38 Mitogen-Activated Protein Kinases/physiology
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