Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmac... more Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE−/−) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4−/−ApoE−/− mice and CCN4+/+ApoE−/− mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4−/−ApoE−/− mice compare...
Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cA... more Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R. Hearts extracted from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocytes isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery, and myocardial injury during reperfusion were evaluated using LDH release and infarct size. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca(2+) transients. Selective activation of either PKA or Epac was found to t...
Arteriosclerosis, thrombosis, and vascular biology, Jul 19, 2016
Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening in coronary ar... more Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening in coronary artery restenosis and vein graft failure, as well as acts as a soil for atherosclerosis. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/TCF signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive,...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2015
Objective— Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, s... more Objective— Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. Approach and Results— Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibr...
Arteriosclerosis, thrombosis, and vascular biology, 2014
Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the athe... more Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of β-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/β-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. Wnt5a induced β-catenin/T-cell factor signaling and retarded oxidative stress (H₂O₂)-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/β-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H₂O₂. Exogenous WISP-1 significantly reduced H₂O₂-induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small int...
Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissu... more Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14(hi) TIMP-3(lo) rabbit foam cells are more invasive and more prone to apoptosis than MMP-14(lo) TIMP-3(hi) cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage ma...
Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarct... more Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachi...
Journal of molecular and cellular cardiology, 2015
Elevation of intracellular cAMP concentration has numerous vascular protective effects that are i... more Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, o...
Arteriosclerosis, thrombosis, and vascular biology, 2014
Our recent studies have highlighted membrane type-1 matrix metalloproteinase (MMP)-14 as a select... more Our recent studies have highlighted membrane type-1 matrix metalloproteinase (MMP)-14 as a selective marker for an invasive subset of macrophages potentially related to atherosclerotic plaque progression. Moreover, colony stimulating factors (CSF) may exert divergent effects on macrophage MMP expression, possibly through microRNAs. We, therefore, aim to identify and test the pathophysiological role of microRNAs, which modulate macrophage MMP-14 expression in atherosclerotic plaque progression. Compared with macrophage CSF-differentiated macrophages, granulocyte/macrophage CSF-matured macrophages exhibited reduced MMP-14 mRNA levels but increased protein expression and activity, which resulted in heightened macrophage invasion. MicroRNA-24, identified to target MMP-14, was accordingly increased in macrophage CSF compared with granulocyte/macrophage CSF macrophages. Silencing microRNA-24 in macrophage CSF macrophages significantly increased MMP-14 expression and enhanced their invasiv...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2011
Objective— Several matrix metalloproteinases (MMPs) have been implicated in extracellular matrix ... more Objective— Several matrix metalloproteinases (MMPs) have been implicated in extracellular matrix destruction and other actions that lead to plaque rupture and myocardial infarction. Conversely, other MMPs have been shown to promote vascular smooth muscle cell (VSMC)–driven neointima formation, which contributes to restenosis, fibrous cap formation, and plaque stability. MMP-3 knockout reduced VSMC accumulation in mouse atherosclerotic plaques, implicating MMP-3 in neointima formation. We therefore investigated the effect of MMP-3 knockout on neointima formation after carotid ligation in vivo and VSMC migration in vitro. Methods and Results— Twenty-eight days after left carotid ligation, MMP-3 knockout significantly reduced neointima formation (75%, P <0.01) compared with wild-type (WT) littermates, and also reduced remodeling of ligated and contralateral carotid arteries. Gelatin zymography illustrated that MMP-3 knockout abolished MMP-9 activation in ligated carotids and scratch...
Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmac... more Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE−/−) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4−/−ApoE−/− mice and CCN4+/+ApoE−/− mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4−/−ApoE−/− mice compare...
Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cA... more Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R. Hearts extracted from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocytes isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery, and myocardial injury during reperfusion were evaluated using LDH release and infarct size. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca(2+) transients. Selective activation of either PKA or Epac was found to t...
Arteriosclerosis, thrombosis, and vascular biology, Jul 19, 2016
Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening in coronary ar... more Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening in coronary artery restenosis and vein graft failure, as well as acts as a soil for atherosclerosis. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/TCF signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive,...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2015
Objective— Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, s... more Objective— Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. Approach and Results— Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibr...
Arteriosclerosis, thrombosis, and vascular biology, 2014
Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the athe... more Apoptosis of vascular smooth muscle cells (VSMCs) contributes to thinning and rupture of the atherosclerotic plaque fibrous cap and is thereby associated with myocardial infarction. Wnt protein activation of β-catenin regulates numerous genes that are associated with cell survival. We therefore investigated Wnt/β-catenin survival signaling in VSMCs and assessed the presence of this pathway in human atherosclerotic plaques at various stages of the disease process. Wnt5a induced β-catenin/T-cell factor signaling and retarded oxidative stress (H₂O₂)-induced apoptosis in mouse aortic VSMCs. Quantification of mRNA levels revealed a >4-fold (P<0.05; n=9) increase in the expression of the Wnt/β-catenin responsive gene, Wnt1-inducible secreted protein-1 (WISP-1), which was dependent on cAMP response element-binding protein and sustained in the presence of H₂O₂. Exogenous WISP-1 significantly reduced H₂O₂-induced apoptosis by 43% (P<0.05; n=3) and was shown using silencing small int...
Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissu... more Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14(hi) TIMP-3(lo) rabbit foam cells are more invasive and more prone to apoptosis than MMP-14(lo) TIMP-3(hi) cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage ma...
Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarct... more Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachi...
Journal of molecular and cellular cardiology, 2015
Elevation of intracellular cAMP concentration has numerous vascular protective effects that are i... more Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, o...
Arteriosclerosis, thrombosis, and vascular biology, 2014
Our recent studies have highlighted membrane type-1 matrix metalloproteinase (MMP)-14 as a select... more Our recent studies have highlighted membrane type-1 matrix metalloproteinase (MMP)-14 as a selective marker for an invasive subset of macrophages potentially related to atherosclerotic plaque progression. Moreover, colony stimulating factors (CSF) may exert divergent effects on macrophage MMP expression, possibly through microRNAs. We, therefore, aim to identify and test the pathophysiological role of microRNAs, which modulate macrophage MMP-14 expression in atherosclerotic plaque progression. Compared with macrophage CSF-differentiated macrophages, granulocyte/macrophage CSF-matured macrophages exhibited reduced MMP-14 mRNA levels but increased protein expression and activity, which resulted in heightened macrophage invasion. MicroRNA-24, identified to target MMP-14, was accordingly increased in macrophage CSF compared with granulocyte/macrophage CSF macrophages. Silencing microRNA-24 in macrophage CSF macrophages significantly increased MMP-14 expression and enhanced their invasiv...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2011
Objective— Several matrix metalloproteinases (MMPs) have been implicated in extracellular matrix ... more Objective— Several matrix metalloproteinases (MMPs) have been implicated in extracellular matrix destruction and other actions that lead to plaque rupture and myocardial infarction. Conversely, other MMPs have been shown to promote vascular smooth muscle cell (VSMC)–driven neointima formation, which contributes to restenosis, fibrous cap formation, and plaque stability. MMP-3 knockout reduced VSMC accumulation in mouse atherosclerotic plaques, implicating MMP-3 in neointima formation. We therefore investigated the effect of MMP-3 knockout on neointima formation after carotid ligation in vivo and VSMC migration in vitro. Methods and Results— Twenty-eight days after left carotid ligation, MMP-3 knockout significantly reduced neointima formation (75%, P <0.01) compared with wild-type (WT) littermates, and also reduced remodeling of ligated and contralateral carotid arteries. Gelatin zymography illustrated that MMP-3 knockout abolished MMP-9 activation in ligated carotids and scratch...
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Papers by Jason Johnson