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1.  Three-dimensional reconstruction of neovasculature in solid tumors and basement membrane matrix using ex vivo X-ray micro-computed tomography 
Objective
To create accurate, high resolution 3D reconstructions of neovasculature structures in xenografted tumors and Matrigel plugs for quantitative analyses in angiogenesis studies in animal models.
Methods
The competent neovasculature within xenografted solid tumors or Matrigel plugs in mice was perfused with Microfil, a radio-opaque, hydrophilic polymerizing contrast agent, by systemic perfusion of the blood circulation via the heart. The perfused tumors and plugs were resected and scanned by X-ray micro-computed tomography to generate stacks of 2D images showing the radio-opaque material. A non-biased, precise post-processing scheme was employed to eliminate background X-ray absorbance from the extra-vascular tissue. The revised binary image stacks were compiled to reveal the Microfil-casted neovasculature as 3D reconstructions. Vascular structural parameters were calculated from the refined 3D reconstructions using the scanner software.
Results
Clarified 3D reconstructions were sufficiently precise to allow measurements of vascular architecture to a diametric limit of resolution of 3 μm in tumors and plugs.
Conclusions
Ex vivo micro-computed tomography can be used for 3D reconstruction and quantitative analysis of neovasculature including microcirculation in solid tumors and Matrigel plugs. This method can be generally applied for reconstructing and measuring vascular structures in 3 dimensions.
doi:10.1111/micc.12102
PMCID: PMC4185970  PMID: 25279426
Micro-computed tomography (microCT); Vascular imaging; Tumor angiogenesis; Matrigel plug; Microvasculature
2.  Attenuation of Experimental Atherosclerosis by Interleukin-19 
Objective
Interleukin-19 (IL-19) is putative Th2, anti-inflammatory interleukin. Its expression in, and potential role in atherogenesis is unknown. IL-19 is not detected in normal artery, and is expressed to a greater degree in plaque from symptomatic vs. asymptomatic patients, suggesting a compensatory-counter regulatory function. We tested if IL-19 could reduce atherosclerosis in susceptible mice, and identified plausible mechanisms.
Approach and Results
LDLR−/− mice fed an atherogenic diet and injected with either 1.0ng/g/day or 10.0ng/g/day rmIL-19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Weight gain, cholesterol and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNγ, IL-1β and IL-12β, and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19 treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated IL-19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells (EC), vascular smooth muscle cells (VSMC), and bone marrow-derived macrophages (BMDM) with IL-19 resulted in a significant decrease in chemokine mRNA, and in the mRNA-stability protein HuR.
Conclusions
These data suggest IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.
doi:10.1161/ATVBAHA.113.301521
PMCID: PMC3950941  PMID: 23950143
atherosclerosis; Interleukin-19; chemokines; macrophage
3.  Myeloid Cell 5-Lipoxygenase Activating Protein Modulates the Response to Vascular Injury 
Circulation research  2012;112(3):432-440.
Rationale
Human genetics have implicated the 5- lipoxygenase (5-LO) enzyme in the pathogenesis of cardiovascular disease and an inhibitor of the 5-LO activating protein (FLAP) is in clinical development for asthma.
Objective
Here we determined whether FLAP deletion modifies the response to vascular injury.
Methods and Results
Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout (FLAP KO) mice and wild type (WT) controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP KOs while endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine (BrdU) incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP KO macrophages was depressed and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B4 (LTB4). FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C (TNC), which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin and upregulation of vascular cell adhesion molecule (VCAM) -1 was also suppressed in FLAP KO mice. Transplantation of FLAP replete myeloid cells rescued the proliferative response to vascular injury.
Conclusion
Expression of lesional FLAP in myeloid cells promotes LTB4 dependent VSMC phenotypic modulation, intimal migration and proliferation.
doi:10.1161/CIRCRESAHA.112.300755
PMCID: PMC3565603  PMID: 23250985
Restenosis; vascular injury; leukotrienes; inflammation; angioplasty and stenting; smooth muscle cell; animal model of human disease remodeling
4.  Vascular COX-2 Modulates Blood Pressure and Thrombosis in Mice 
Science translational medicine  2012;4(132):10.1126/scitranslmed.3003787.
Prostacyclin (PGI2) is a vasodilator and platelet inhibitor, properties consistent with cardioprotection. More than a decade ago, inhibition of cyclooxygenase-2 (COX-2) by the nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib and celecoxib was found to reduce the amount of the major metabolite of PGI2 (PGI-M) in the urine of healthy volunteers. This suggested that NSAIDs might cause adverse cardiovascular events by reducing production of cardioprotective PGI2. This prediction was based on the assumption that the concentration of PGI-M in urine likely reflected vascular production of PGI2 and that other cardioprotective mediators, especially nitric oxide (NO), were not able to compensate for the loss of PGI2. Subsequently, eight placebo-controlled clinical trials showed that NSAIDs that block COX-2 increase adverse cardiovascular events. We connect tissue-specific effects of NSAID action and functional correlates in mice with clinical outcomes in humans by showing that deletion of COX-2 in the mouse vasculature reduces excretion of PGI-M in urine and predisposes the animals to both hypertension and thrombosis. Furthermore, vascular disruption of COX-2 depressed expression of endothelial NO synthase and the consequent release and function of NO. Thus, suppression of PGI2 formation resulting from deletion of vascular COX-2 is sufficient to explain the cardiovascular hazard from NSAIDs, which is likely to be augmented by secondary mechanisms such as suppression of NO production.
doi:10.1126/scitranslmed.3003787
PMCID: PMC3882087  PMID: 22553252
5.  Inhibition of Rho-kinase attenuates endothelial–leukocyte interaction during ischemia–reperfusion injury 
Resuscitation from hemorrhagic shock induces endothelial dysfunction and activates inflammatory cascades leading to organ damage. Following restoration of blood flow to ischemic vascular beds, leukocyte–endothelium interactions leading to leukocyte infiltration into the vascular wall occur very early due, in part, to the loss of endothelium-derived nitric oxide (NO). The mechanism by which ischemia–reperfusion injury impairs endothelium-derived NO is not completely understood. We hypothesized that inhibition of Rho-kinase could exert beneficial effects following hemorrhagic shock by preserving endothelial function and attenuating leukocyte trafficking in the microcirculation. Using intravital microscopy, we found that resuscitation from hemorrhage acutely increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. Treatment of mice with the Rho-kinase inhibitor fasudil, markedly attenuated leukocyte–endothelium interaction in response to hemorrhage/reinfusion. The beneficial effect of fasudil was not observed in endothelial nitric oxide synthase (eNOS)−/− mice. In conclusion, inhibition of Rho-kinase prevents inflammatory leukocyte trafficking in the microcirculation via an eNOS-dependent mechanism. Our data support a role for Rho-kinase inhibitors in the treatment of ischemia–reperfusion injury.
doi:10.1177/1358863X12459790
PMCID: PMC3807093  PMID: 23015643
hemorrhage/reperfusion injury; inflammation; microcirculation; nitric oxide; Rho-kinase
6.  PKC Upregulates ICAM-1 And Leukocyte-Endothelium Interactions in Hyperglycemia Via Activation of Endothelial Expressed Calpain 
Objective
We tested the hypothesis of a role for the calcium-dependent protease calpain in the endothelial dysfunction induced by hyperglycemic activation of protein kinase C (PKC).
Methods and Results
Chronic hyperglycemia with insulin deficiency (Type-1 diabetes) was induced in rats by streptozotocin. Total PKC and calpain activities, along with activity and expression level of the two endothelial-expressed calpains isoforms, µ- and m-calpain, were measured in vascular tissue homogenates by enzymatic assays and western blot analysis, respectively. Intravital microscopy was used to measure and correlate leukocyte-endothelium interactions with calpain activity in the microcirculation. Expression levels and endothelial localization of the inflammatory adhesion molecule ICAM-1 were studied by western blot analysis and immunofluorescence, respectively. The mechanistic role of hyperglycemia alone in the process of PKC-induced calpain activation and actions was also investigated. We found that in the Type-1 diabetic vasculature PKC selectively upregulates the activity of the µ-calpain isoform. Mechanistic studies confirmed a role for hyperglycemia and PKCβ in this process. The functional implications of PKC-induced calpain activation were upregulation of endothelial expressed ICAM-1 and leukocyte-endothelium interactions.
Conclusions
Our results uncover the role of µ-calpain in the endothelial dysfunction of PKC. Calpain may represent a novel molecular target for the treatment of PKC-associated diabetic vascular disease.
doi:10.1161/ATVBAHA.110.217901
PMCID: PMC3086836  PMID: 21071702
calpain; PKC; diabetes; vascular complications; leukocytes; endothelium
7.  Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice 
Cell metabolism  2010;11(5):379-389.
Summary
To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls, but reduced to below control values by a VCAM-1 blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.
doi:10.1016/j.cmet.2010.03.013
PMCID: PMC3020149  PMID: 20444418
8.  Protective vascular and myocardial effects of adiponectin 
SUMMARY
Adiponectin is an abundant plasma protein secreted from adipocytes that elicits protective effects in the vasculature and myocardium. In obesity and insulin-resistant states, adiponectin levels are reduced and loss of its protective effects might contribute to the excess cardiovascular risk observed in these conditions. Adiponectin ameliorates the progression of macrovascular disease in rodent models, consistent with its correlation with improved vascular outcomes in epidemiological studies. The mechanisms of adiponectin signaling are multiple and vary among its cellular sites of action. In endothelial cells, adiponectin enhances production of nitric oxide, suppresses production of reactive oxygen species, and protects cells from inflammation that results from exposure to high glucose levels or tumor necrosis factor, through activation of AMP-activated protein kinase and cyclic AMP-dependent protein kinase (also known as protein kinase A) signaling cascades. In the myocardium, adiponectin-mediated protection from ischemia-reperfusion injury is linked to cyclo-oxygenase-2-mediated suppression of tumor necrosis factor signaling, inhibition of apoptosis by AMP-activated protein kinase, and inhibition of excess peroxynitrite-induced oxidative and nitrative stress. In this Review, we provide an update of studies of the signaling effects of adiponectin in endothelial cells and cardiomyocytes.
doi:10.1038/ncpcardio1398
PMCID: PMC2658652  PMID: 19029992
atherosclerosis; endothelial function; hyperglycemia; ischemia-reperfusion; oxidative stress
9.  Inhibition of Rho-Kinase Leads to Rapid Activation of Phosphatidylinositol 3-Kinase/Protein Kinase Akt and Cardiovascular Protection 
Objective
Rho-Kinase activity is increased in cardiovascular diseases and in patients with cardiovascular risk factors. However, it is not known whether inhibition of Rho-kinase could lead to cardiovascular protection and, if so, by what mechanism.
Methods and Results
In human endothelial cells, the Rho-kinase inhibitor, hydroxyfasudil (HF) (1 to 100 μmol/L), increased Akt serine-473 phosphorylation within 15 minutes, leading to a 2.2-fold and 4.0-fold increase in Akt kinase activity and nitric oxide (NO) release, respectively. Activation of Akt and eNOS by HF was completely blocked by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002 (10 μmol/L). To determine the physiological relevance of this pathway, we used 2 models of ischemia-reperfusion (I/R) injury. Acute administration of fasudil (10 mg/kg, intraperitoneal, 1 hour before ischemia) decreased leukocyte recruitment and adhesion to the mesenteric endothelium after I/R injury in wild-type but not eNOS−/− mice. Similarly, treatment with fasudil decreased myocardial infarct size by 38% in rats subjected to transient coronary artery occlusion. Cotreatment with 2 PI3-kinase inhibitors, wortmannin and LY294002, or the eNOS inhibitor, L-NAME, blocked the cardiovascular protective effects of fasudil.
Conclusions
Inhibition of Rho-kinase leads to the activation of the PI3-kinase/Akt/eNOS pathway and cardiovascular protection. These findings suggest that Rho-kinase may play an important role in mediating the inflammatory response to I/R injury.
doi:10.1161/01.ATV.0000142813.33538.82
PMCID: PMC2649731  PMID: 15319269
endothelium; inflammation; infarction; nitric oxide; ischemia-reperfusion
10.  Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo 
Journal of Clinical Investigation  2007;117(6):1718-1726.
This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad–/–) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad–/– mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad–/– mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-α–induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with Nω-nitro- l-arginine methyl ester ( l-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.
doi:10.1172/JCI29623
PMCID: PMC1878515  PMID: 17549259
11.  Essential Role of P-Selectin in the Initiation of the Inflammatory Response Induced by Hemorrhage and Reinfusion  
Resuscitation from hemorrhage induces profound pathophysiologic alterations and activates inflammatory cascades able to initiate neutrophil accumulation in a variety of tissues. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that significant leukocyte–endothelium interactions occur very early in other forms of ischemia/reperfusion (i.e., splanchnic ischemia/reperfusion and traumatic shock) which are largely mediated by increased expression of the adhesion molecule, P-selectin, on the vascular endothelium. Here we postulated that increased endothelial expression of P-selectin in the microvasculature would play an essential role in initiating the inflammatory signaling of hemorrhagic shock. Using intravital microscopy, we found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti–P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-1 immunoglobulin, exhibited markedly attenuated leukocyte–endothelium interaction after hemorrhagic shock. Thus, activation of P-selectin protein on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Moreover, endogenous levels of PSGL-1 mRNA were significantly increased in the lung, liver, and small intestine of wild-type mice subjected to hemorrhagic shock. Since PSGL-1 promotes adhesive interactions largely through P-selectin expressed on the vascular endothelium, this result further supports the crucial role played by P-selectin in the recruitment of leukocytes during hemorrhagic shock.
PMCID: PMC2193039  PMID: 10075976
mRNA; leukocyte; endothelium; immunohistochemistry; intravital microscopy

Results 1-11 (11)