vasodilation-isolated-vessels-isolation-extracellular-matrix-tight

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Medical College of Wisconsin

Development of coronary collateral vessels is impaired in patients with diabetes mellitus. We tested the hypothesis that hyperglycemia alone attenuates collateral development and abolishes proliferative properties of myocardial interstitial fluid (MIF) by enhancing expression of matrix metalloproteinases (MMP) and angiostatin.

Chronic hyperglycemia attenuates coronary collateral development and impairs proliferative properties of myocardial interstitial fluid by production of angiostatin.

Coronary collateral development relies on the coordinated secretion of growth factors. However, alone they are insufficient for permanent collateral growth. We utilized proteomics to identify other important proteins in the extracellular environment that could facilitate collateralization. Chronically instrumented dogs developed coronary collaterals by the repetitive occlusion method. Subendocardial (0.19 +/- 0.04, 0.27 +/- 0.06, 0.48 +/- 0.10, and 0.81 +/- 0.11 ml x min(-1) x g(-1) on days 1, 7, 14, and 21, respectively) and subepicardial (0.14 +/- 0.01, 0.36 +/- 0.06, 0.51 +/- 0.07, and 0.71 +/- 0.08 ml x min(-1) x g(-1) on days 1, 7, 14, and 21, respectively) blood flow increased in animals subjected to repetitive occlusion. Sham animals exhibited no changes in blood flow. Myocardial interstitial fluid (MIF) from both groups was analyzed by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization time-of-flight identification. The acute-phase protein haptoglobin was identified in the group subjected to repetitive occlusion. ELISA of MIF showed haptoglobin to be elevated at all time points of collateral development compared with sham, with maximal production on day 7. Purified haptoglobin dose dependently stimulated endothelial cells to form tubes and vascular smooth muscle cells to migrate. Purified haptoglobin did not stimulate proliferation of either cell type. The relative contribution of haptoglobin to the chemotactic properties of MIF was tested using a neutralizing antibody. Neutralized MIF could not stimulate smooth muscle cells to migrate at any time during collateral development. Endothelial cell tube formation was inhibited after the midpoint of collateralization. Therefore, the acute-phase protein haptoglobin plays a critical role during coronary collateralization.

Haptoglobin expression and activity during coronary collateralization.

Nitric oxide is an important messenger in numerous biological processes, such as angiogenesis, hypoxic vasodilation, and cardioprotection. Although nitric oxide synthases (NOS) produce the bulk of NO, there is increasing interest in NOS independent generation of NO in vivo, particularly during hypoxia or anoxia, where low oxygen tensions limit NOS activity. Interventions that can increase NO bioavailability have significant therapeutic potential. The use of far red and near infrared light (R/NIR) can reduce infarct size, protect neurons from methanol toxicity, and stimulate angiogenesis. How R/NIR modulates these processes in vivo and in vitro is unknown, but it has been suggested that increases in NO levels are involved. In this study we examined if R/NIR light could facilitate the release of NO from nitrosyl heme proteins. In addition, we examined if R/NIR light could enhance the protective effects of nitrite on ischemia and reperfusion injury in the rabbit heart. We show both in purified systems and in myocardium that R/NIR light can decay nitrosyl hemes and release NO, and that this released NO may enhance the cardioprotective effects of nitrite. Thus, the photodissociation to NO and its synergistic effect with sodium nitrite may represent a noninvasive and site-specific means for increasing NO bioavailability.

Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: potential role in cardioprotection.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis.

Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles.

Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: implications for preconditioning.

The tight skin mouse (Tsk(-/+)) is a model of scleroderma characterized by impaired vasoreactivity, increased oxidative stress, attenuated angiogenic response to VEGF and production of the angiogenesis inhibitor angiostatin. Low-level light therapy (LLLT) stimulates angiogenesis in myocardial infarction and chemotherapy-induced mucositis. We hypothesize that repetitive LLLT restores vessel growth in the ischemic hindlimb of Tsk(-/+) mice by attenuating angiostatin and enhancing angiomotin effects in vivo. C57Bl/6J and Tsk(-/+) mice underwent ligation of the femoral artery. Relative blood flow to the foot was measured using a laser Doppler imager. Tsk(-/+) mice received LLLT (670 nm, 50 mW cm(-2), 30 J cm(-2)) for 10 min per day for 14 days. Vascular density was determined using lycopersicom lectin staining. Immunofluorescent labeling, Western blot analysis and immunoprecipitation were used to determine angiostatin and angiomotin expression. Recovery of blood flow to the ischemic limb was reduced in Tsk(-/+) compared with C57Bl/6 mice 2 weeks after surgery. LLLT treatment of Tsk(-/+) mice restored blood flow to levels observed in C57Bl/6 mice. Vascular density was decreased, angiostatin expression was enhanced and angiomotin depressed in the ischemic hindlimb of Tsk(-/+) mice. LLLT treatment reversed these abnormalities. LLLT stimulates angiogenesis by increasing angiomotin and decreasing angiostatin expression in the ischemic hindlimb of Tsk(-/+) mice.

Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse.

Nitric oxide (NO) is a crucial mediator of hindlimb collateralization and angiogenesis. Within tissues there are nitrosyl-heme proteins which have the potential to generate NO under conditions of hypoxia or low pH. Low level irradiation of blood and muscle with light in the far red/near infrared spectrum (670 nm, R/NIR) facilitates NO release. Therefore, we assessed the impact of red light exposure on the stimulation of femoral artery collateralization. Rabbits and mice underwent unilateral resection of the femoral artery and chronic R/NIR treatment. The direct NO scavenger carboxy-PTIO and the nitric oxide synthase (NOS) inhibitor L-NAME were also administered in the presence of R/NIR. DAF fluorescence assessed R/NIR changes in NO levels within endothelial cells. In vitro measures of R/NIR induced angiogenesis were assessed by endothelial cell proliferation and migration. R/NIR significantly increased collateral vessel number which could not be attenuated with L-NAME. R/NIR induced collateralization was abolished with c-PTIO. In vitro, NO production increased in endothelial cells with R/NIR exposure, and this finding was independent of NOS inhibition. Similarly R/NIR induced proliferation and tube formation in a NO dependent manner. Finally, nitrite supplementation accelerated R/NIR collateralization in wild type C57Bl/6 mice. In an eNOS deficient transgenic mouse model, R/NIR restores collateral development. In conclusion, R/NIR increases NO levels independent of NOS activity, and leads to the observed enhancement of hindlimb collateralization.

Far red/near infrared light treatment promotes femoral artery collateralization in the ischemic hindlimb.

Collateral development: the quest continues.

Interferon regulatory factor 5 (IRF5) has been called a "master switch" for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0-100 μg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50μg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

Article	Year
Chronic hyperglycemia attenuates coronary collateral development and impairs proliferative properties of myocardial interstitial fluid by production of angiostatin. Circulation\| PubMed ID: 15136506	2004
Haptoglobin expression and activity during coronary collateralization. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 15550518	2005
Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: potential role in cardioprotection. Journal of molecular and cellular cardiology\| PubMed ID: 19328206	2009
Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis. American journal of physiology. Cell physiology\| PubMed ID: 21160034	2011
Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: implications for preconditioning. Anesthesiology\| PubMed ID: 21862887	2011
Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse. Photochemistry and photobiology\| PubMed ID: 23231468	2013
Far red/near infrared light treatment promotes femoral artery collateralization in the ischemic hindlimb. Journal of molecular and cellular cardiology\| PubMed ID: 23702287	2013
Collateral development: the quest continues. Circulation research\| PubMed ID: 24526671	2014
An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice. PloS one\| PubMed ID: 27050551	2016

Nicole L. Lohr

Biography

JoVE Journal Publications

Other Publications