We have shown that nanoparticle inhalation impairs endothelium-dependent vasodilation in coronary arterioles. It is unknown whether local reactive oxygen species (ROS) contribute to this effect. Rats were exposed to TiO 2 nanoparticles via inhalation to produce a pulmonary deposition of 10 µg. Coronary arterioles were isolated from the left anterior descending artery distribution, and responses to acetylcholine, arachidonic acid, and U46619 were assessed. Contributions of nitric oxide synthase and prostaglandin were assessed via competitive inhibition with N G -Monomethyl-L-Arginine (L-NMMA) and indomethacin. Microvascular wall ROS were quantified via dihydroethidium (DHE) fluorescence. Coronary arterioles from rats exposed to nano-TiO 2 exhibited an attenuated vasodilator response to ACh, and this coincided with a 45% increase in DHE fluorescence. Coincubation with 2,2,6,6-tetramethylpiperidine-N-oxyl and catalase ameliorated impairments in ACh-induced vasodilation from nanoparticle exposed rats. Incubation with either L-NMMA or indomethacin significantly attenuated Ach-induced vasodilation in sham-control rats, but had no effect in rats exposed to nano-TiO 2 . Arachidonic acid induced vasoconstriction in coronary arterioles from rats exposed to nano-TiO 2 , but dilated arterioles from sham-control rats. These results suggest that nanoparticle exposure significantly impairs endothelium-dependent vasoreactivity in coronary arterioles, and this may be due in large part to increases in microvascular ROS. Furthermore, altered prostanoid formation may also contribute to this dysfunction. Such disturbances in coronary microvascular function may contribute to the cardiac events associated with exposure to particles in this size range.

Exposure to fine particulate matter (PM, mean aerodynamic diameter ≤ 2.5 μm) has been shown to be a risk factor for cardiovascular disease mortality and may contribute to acute coronary events such as myocardial infarction (MI). There is sufficient reason to believe that smaller particles, such as nanoparticles, might be even more detrimental than larger-sized particles due to their increased surface area and higher pulmonary deposition. Our lab showed that nanoparticle inhalation impairs endothelium-dependent arteriolar vasodilation in skeletal muscle. However, it is not known if coronary microvascular endothelial function is affected in a similar manner. Rats were exposed to filtered air (control) or TiO2 nanoparticles (primary particle diameter, ~21 nm) via inhalation at concentrations that produced measured depositions (10 μg) relevant to ambient air pollution. Subepicardial arterioles (~150 μm in diameter) were isolated and responses to transmural pressure, flow-induced dilation (FID), acetylcholine, the Ca2+ ionophore A23187, and sodium nitroprusside (SNP) assessed. Myogenic responsiveness was preserved between groups. In addition, there was no difference in the vasodilation to SNP, signifying that smooth muscle sensitivity to nitric oxide (NO) is unaffected by nano-TiO2 exposure. However, inhalation of nano-TiO2 produced an increase in spontaneous tone in coronary arterioles and also impaired endothelium-dependent FID. In addition, ACh- and A23187-induced vasodilation was also blunted in arterioles after inhalation of nano-TiO2. Data showed that nanoparticle exposure significantly impairs endothelium-dependent vasodilation in subepicardial arterioles. Such disturbances in coronary microvascular function are consistent with the cardiac events associated with particle pollution exposure.

The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms.

October 7, 2009; doi:10.1152/ajpregu.00178.2009.-The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flowinduced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized ϩ estrogen replaced (OVE) female Fischer-344 rats to assess flowinduced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 Ϯ 4 vs. old: 34 Ϯ 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by ϳ160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with N G -nitro-L-arginine methyl ester (L-NAME), declined with age. L-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, L-NAME reduced flowinduced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flowinduced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age.endothelial nitric oxide synthase; Akt; nitric oxide; ovariectomy THE RISK FOR CARDIOVASCULAR disease (CVD) and heart failure increase with advancing age; however, sexual dimorphism exists in the chronological development of these risks (22,47). Although the chronological rate of aging is independent of sex, mechanisms that regulate the cardiovascular system across the lifespan may differ dramatically between men and women. The risk for CVD in men begins to increase at approximately the same age that flow-mediated vasodilation begins to decline (5). Women also exhibit this age-related impairment of flow-mediated vasodilation; however, significant reduction of flow-mediated dilation becomes apparent at the age of menopause, more than a decade later than in men (5). The cardioprotection that women experience until menopause is presumably due to the presence of ovarian estrogen and results in a sex-related delay of the expression of CVD (49). Chronic estrogen treatment has been shown to enhance endothelial function in a number of vascular beds (27, 31, 39), in part, through a pathway involving activation of Akt/PKB and subsequent phosphorylation of endothelial nitric oxide synthase (eNOS) (3,10,15,43,44). Endothelium-dependent vasodilation to a...

Vascular compromise and the accompanying perfusion deficits cause or complicate a large array of disease conditions and treatment failures. This has prompted the exploration of therapeutic strategies to repair or regenerate vasculatures thereby establishing more competent microcirculatory beds. Growing evidence indicates that an increase in vessel numbers within a tissue does not necessarily promote an increase in tissue perfusion. Effective regeneration of a microcirculation entails the integration of new stable microvessel segments into the network via neovascularization. Beginning with angiogenesis, neovascularization entails an integrated series of vascular activities leading to the formation of a new mature microcirculation and includes vascular guidance and inosculation, vessel maturation, pruning, arterio-venous specification, network patterning, structural adaptation, intussusception, and microvascular stabilization. While the generation of new vessel segments is necessary to expand a network, without the concomitant neovessel remodeling and adaptation processes intrinsic to microvascular network formation, these additional vessel segments give rise to a dysfunctional microcirculation. While many of the mechanisms regulating angiogenesis have been detailed, a thorough understanding of the mechanisms driving post-angiogenesis activities specific to neovascularization has yet to be fully realized, but is necessary in order to develop effective therapeutic strategies for repairing compromised microcirculations as a means to treat disease.

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P

A three-dimensional tissue construct was created using adipose-derived stromal vascular fraction (SVF) cells and evaluated as a microvascular protection treatment in a myocardial infarction (MI) model. This study evaluated coronary blood flow (BF) and global left ventricular function after MI with and without the SVF construct. Fischer-344 rats were separated into four groups: sham operation (sham), MI, MI Vicryl patch (no cells), and MI SVF construct (MI SVF). SVF cells were labeled with green fluorescent protein (GFP). Immediately postinfarct, constructs were implanted onto the epicardium at the site of ischemia. Four weeks postsurgery, the coronary BF reserve was significantly decreased by 67% in the MI group and 75% in the MI Vicryl group compared with the sham group. The coronary BF reserve of the sham and MI SVF groups in the area at risk was not significantly different (sham group: 83 ± 22% and MI SVF group: 57 ± 22%). Griffonia simplicifolia I and GFP-positive SVF immunostaining revealed engrafted SVF cells around microvessels in the infarct region 4 wk postimplant. Overall heart function, specifically ejection fraction, was significantly greater in MI SVF hearts compared with MI and MI Vicryl hearts (MI SVF: 66 ± 4%, MI: 37 ± 8%, and MI Vicryl: 29 ± 6%). In conclusion, adipose-derived SVF cells can be used to construct a novel therapeutic modality for treating microvascular instability and ischemia through implantation on the epicardial surface of the heart. The SVF construct implanted immediately after MI not only maintains heart function but also sustains microvascular perfusion and function in the infarct area by sustaining the coronary BF reserve.

Kang LS, Chen B, Reyes RA, LeBlanc AJ, Teng B, Mustafa SJ, Muller-Delp JM. Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles. Am J Physiol Heart Circ Physiol 300: H2105-H2115, 2011. First published March 25, 2011 doi:10.1152/ajpheart.00349.2010.-Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O 2 Ϫ and H2O2 in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX ϩ estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flowinduced NO production, whereas flow-induced H 2O2 production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 M) or the H2O2 scavenger catalase (100 U/ml). Removal of H 2O2 with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O 2 Ϫ and H2O2 are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O 2 Ϫ buffering by SOD contributes to impaired flowinduced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O 2 Ϫ homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age. superoxide dismutase; hydrogen peroxide; ovariectomy; estrogen replacement