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Background: Fibrinogen (Fgn) is one of several pathogenic factors in the brain microvasculature. It is upregulated in the blood of the aged, Alzheimer’s disease, and other neurological disease patients. We explored the possible role of a human serum Fgn (hFgn) mediated mechanism for endothelial and blood-brain barrier (BBB) dysregulation in primary human brain microvascular endothelial cells (HBMEC). Methods: To assess the endothelial and BBB dysregulation ex vivo, we measured protein expression change by western blot and immunofluorescence in hFgn-treated HBMEC. Cell viability was determined by NAD(P)H-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, and in vitro BBB-permeability was assessed by transwell migration assay. Stable DRP1 knockdown cells were developed by transduction of shRNA(h) lentivirus particles in HBMEC. Results: Clinical studies have reported that Fgn is an abundant protein in human blood plasma at concentrations ranging from 1.5–4 mg/mL with a normal half-life of 3–5 days, and the plasma concentration of Fgn increased progressively with age in healthy subjects. We observed that 4 mg/mL of hFgn induced significant cytotoxicity and generated mitochondrial reactive oxygen species in HBMEC within 24 h. With the same concentration of Fgn, we observed that most of the mitochondria were elongated or hyperfused, possibly due to significant reduction of phosphorylated dynamin related protein 1 (pDRP1[S616]) in hFgn-treated HBMEC. Moreover, mitochondrial oxidative phosphorylation (OXPHOS)-related mitochondrial complex-II, -III, and -V proteins were also decreased in hFgn-treated HBMEC. Interestingly, autophagy-related, microtubule-associated protein light chain 3B-II (LC3B-II) and lysosomal membrane-associated protein 2 (LAMP2) decreased, while a negative regulator of autophagy, p62, increased in hFgn-treated HBMEC, suggesting hFgn-mediated induction of defective autophagy. Importantly, hFgn induced BBB permeability, possibly due to loss of several BBB-related proteins such as JAM-A, occludin, ZO-1/-2, and PECAM-1 in HBMEC. DRP1 knockdown by shRNA(h) resulted in reduction of OXPHOS and BBB-related proteins as well as defective autophagy induction in HBMEC. Conclusions: Our results suggest that detrimental effects of elevated Fgn downregulates DRP1, leading to endothelial and BBB dysfunction in the brain microvasculature, which affects neuronal health/function and adds vulnerability to strokes and neurological diseases. HL148836, AG063345, NS114286, AG047296, and AG074489. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging-associated structural alterations and functional decline of the brain microvasculature contribute to the development of cognitive impairment and dementias such as Alzheimer’s disease. The mitochondrial loss and dysfunction appear to play a major role in mechanisms leading to aging-associated changes of the microvasculature, including increased blood brain barrier permeability, vascular rarefication, or decreased branching. SS-31, a small, water-soluble tetra-peptide has been shown to be effective in improving the aging phenotype in healthy, aged rodents as well as in models of neurodegenerative disease. However, the beneficial effects of SS-31 have not been examined on the brain microvascular proteome.We tested the hypothesis that targeting mitochondria with SS-31 rescues aging-associated changes in the brain microvascular proteome.Aged male (> 18 months) C57Bl6/J mice were randomly assigned to the SS-31 treatment (10 mg/kg; i.p.; n = 5) or vehicle saline (n = 5) groups. Mice were treated daily for two weeks. Cerebral blood flow (CBF) was measured using laser speckle imaging at four time points (0, 3, 7, 14 days). Then, cortical microvessels (MVs) containing a mixture of end arterioles, capillaries, and venules, < 70mm, were isolated and used for Orbitrap Eclipse Tribrid mass spectrometry.During the two-week period, baseline CBF was similar between SS-31 and vehicle groups. Bioinformatic analysis showed that treatment with SS-31 resulted in significant differences in protein abundance of cortical MVs vs. vehicle. Twelve percent (107) of the identified 6,266 proteins were mitochondria associated (oxidative phosphorylation, metabolism, the antioxidant defense system) and mitochondrial dynamics were significantly differentially expressed, Our findings suggest that mitochondria are potential therapeutic targets in the aging brain microvasculature, and that changes in the proteome may contribute to the beneficial effects of SS-31 in aging. Our results warrant further study with the inclusion of female animals to investigate the role of sex-differences in the effects of SS-31. This research was supported in part by: IDeA National Resource for Quantitative Proteomics Voucher Program (R24GM137786), Tulane Brain Institute Research Fund Award to IR, NIH HL-148836, NIH AG-063345, NIH AG-075988, the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program to Dr. Busija, P30GM145498, U54 GM104940. The University of Arkansas for Medical Sciences Bioinformatics Core Facility is supported by the Winthrop P. Rockefeller Cancer Institute and National Institutes of Health grant P20GM121293. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Despite recent therapeutic advancements, ischemic stroke remains a major cause of death and disability. It has been previously demonstrated that ~ 85-kDa recombinant human perlecan domain V (rhPDV) binds to upregulated integrin receptors (α2β1 and α5β1) associated with neuroprotective and functional improvements in various animal models of acute ischemic stroke. Recombinant human perlecan laminin-like globular domain 3 (rhPDVLG3), a 21-kDa C-terminal subdomain of rhPDV, has been demonstrated to more avidly bind to the α2β1 integrin receptor than its parent molecule and consequently was postulated to evoke significant neuroprotective and functional effects. To test this hypothesis, fifty male C57Bl/6 J mice studied in a t-MCAO model were randomly allocated to either rhPDV treatment, rhPDVLG3, or equivalent volume of PBS at the time of reperfusion in a study where all procedures and analyses were conducted blind to treatment. On post-MCAO day 7, 2,3,5-triphenyltetrazolium chloride staining of brain slices was used to quantify infarct volume. We observed that treatment with rhPDVLG3 reduced infarct volume by 65.6% (p = 0.0001), improved weight loss (p p LG3 was observed to significantly reduce mortality due to stroke in one model, an outcome not previously observed for rhPDV. Our initial findings suggest that treatment with rhPDVLG3 provides significant improvement in neuroprotective and functional outcomes in experimental stroke models and that further investigation of rhPDVLG3 as a novel neuroprotective therapy for patients with stroke is warranted.

Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p

Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice. NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V’s effects were inhibited by blockade of α2β1 integrin, suggesting the importance of α2β1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2β1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window. Electronic supplementary material The online version of this article (10.1007/s12975-020-00800-5) contains supplementary material, which is available to authorized users.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca

Differentially expressed (DE) proteins in the cortical microvessels (MVs) of young, middle-aged, and old male and female mice were evaluated using discovery-based proteomics analysis (> 4,200 quantified proteins/group). Most DE proteins (> 90%) showed no significant differences between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from young (8.3%), to middle-aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age-dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase-1/-2, catalase and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre-mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance of these proteins decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial complexes I–V were reduced in old mice, six of these proteins showed a significant reduction in middle-aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age. We suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity.

SARS‐CoV‐2 infection impacts multiple organ systems, including the central nervous system (CNS). Multiple reports have described a variety of neurological manifestations associated with infection that may contribute to worsening COVID‐19. The neuropathology of SARS‐CoV‐2 is not well understood, necessitating the development of relevant animal models for investigation. Here, we report marked neuropathology but with limited virus in the CNS of two non‐human primate models (NHPs) of SARS‐CoV‐2 infection. Adult male and female purpose‐bred Rhesus macaques (RMs; n = 4) and wild‐caught African green monkeys (AGMs; n = 4) were inoculated with the 2019‐nCoV/USA‐WA1/2020 strain of SARS‐CoV‐2 via multi‐route mucosal or aerosol challenge. SARS‐CoV‐2 nucleocapsid (SARS‐N) mRNA was detected in nasal swabs within the first week of inoculation, demonstrating infection of all study animals. All animals were euthanized at the study endpoint of 4 weeks post‐inoculation, with the exception of two AGMs that reached humane endpoints at 8‐ and 22‐days post‐challenge. Seven regions of the CNS were investigated for pathology and virus infection. Archival brain tissues from two non‐infected adult female RMs were used as aged‐matched controls. Mild, but chronic, hypoxemia with impaired gas exchange were suggested by SpO2 values that stayed at or below 95% and elevated blood CO2 in the majority of the study animals. Neuroinflammation was seen throughout the brain and brainstem but with limited virus detection by immunohistochemistry and RNAscope of fixed tissues and viral RNA detection using a highly sensitive CRISPR‐fluorescent detection system on RNA extracted from sectioned brain lysates. In addition, neuronal injury and death were suggested by pyknotic and karyolytic nuclei and cellular blebbing. Limited myelin vacuolation was revealed in two infected animals through Luxol Fast Blue staining. Neuronal cleaved caspase 3 positivity was seen at a greater frequency in infected animals compared to controls, suggesting increased apoptosis in infection. Microhemorrhages were larger and more frequent among infected NHPs, as compared to controls. Neuroinflammation, neuronal injury and death, and microhemorrhages were seen in animals that did not develop severe respiratory disease and may suggest neuropathology contributes to on‐going symptoms of convalesced patients. Our findings in NHPs are in agreement with human autopsy and neuroimaging studies and demonstrate this is a relevant animal model for investigating neuropathological changes associated with COVID‐19. Our results also suggest that hypoxic‐ischemic events leading to energy failure and neuronal injury, contribute to the neuropathological consequences of COVID‐19. Further studies are warranted to elucidate the mechanisms of SARS‐CoV‐2 neuropathogenesis.

Despite promising advancements, ischemic stroke remains a leading cause of death and disability. We previously determined that human recombinant ~85-kDa C-terminal protein fragment of the vascular basement membrane proteoglycan perlecan termed domain V (DV) is neuroprotective, pro-angiogenic, neurogenic and functionally restorative when administered after experimental stroke (transient and permanent middle cerebral artery occlusion -MCAO) in mice and rats. As previous studies suggest, the 23-kDa subdomain of DV called laminin globular domain 3 (LG3) may confer most of DV’s biological activity and is more amenable to production, we tested whether human recombinant perlecan DV LG3 (also termed “DV LG3 ”) might be neuroprotective and functionally restorative in the transient MCAO (intraluminal filament model). Methods: Fifty male mice (C57BL/6J, 10-12 weeks old, 24–30 g) underwent MCAO for 60 min and were survived for seven days. At reperfusion mice were randomly allocated to one of three treatment groups and immediately received either human recombinant DV treatment (positive control, n=16 2 mg/kg, i.p.), LG3 (n=19 6 mg/kg, i.p.), or equivalent volume of PBS vehicle (n=15) ; and again on post-MCAO days 2 and 4. The DV:LG3 dose ratio was selected based on a preliminary in vivo dose evaluation study of therapeutic equivalence. Functional outcome measures (grip strength and grid-hang tests) were determined at baseline and on post-MCAO days 1, 3 and 7. Body weight was measured daily as per welfare. To determine infarct size on post-MCAO day 7, whole brain samples were harvested, sectioned and stained with 2,3,5- Triphenyltetrazolium chloride. The induction of MCAO, DV, DV LG3 or PBS treatments, functional tests and analyses were performed, blindly, by different individuals. While DV treatment significantly reduced mean infarct volume as expected (pLG3 further reduced infarct volumes (pLG3 is neuroprotective and functionally restorative, may be even more effective than DV, and is worthy of more study as a readily producible therapy for ischemic stroke.

Neurological manifestations are a significant complication of coronavirus infection disease-19 (COVID-19). Understanding how COVID-19 contributes to neurological disease is needed for appropriate treatment of infected patients, as well as in initiating relevant follow-up care after recovery. Investigation of autopsied brain tissue has been key to advancing our understanding of the neuropathogenesis of a large number of infectious and non-infectious diseases affecting the central nervous system (CNS). Due to the highly infectious nature of the etiologic agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a paucity of tissues available for comprehensive investigation. Here, we show for the first time, microhemorrhages and neuropathology that is consistent with hypoxic injury in SARS-CoV-2 infected non-human primates (NHPs). Importantly, this was seen among infected animals that did not develop severe respiratory disease. This finding underscores the importance of vaccinating against SARS-CoV-2, even among populations that have a reduced risk for developing of severe disease, to prevent long-term or permanent neurological sequelae. Sparse virus was detected in brain endothelial cells but did not associate with the severity of CNS injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

Sex differences in mitochondrial numbers and function are present in large cerebral arteries, but it is unclear whether these differences extend to the microcirculation. We performed an assessment of mitochondria-related proteins in cerebral microvessels (MVs) isolated from young, male and female, Sprague-Dawley rats. MVs composed of arterioles, capillaries, and venules were isolated from the cerebrum and used to perform a 3 versus 3 quantitative, multiplexed proteomics experiment utilizing tandem mass tags (TMT), coupled with liquid chromatography/mass spectrometry (LC/MS). MS data and bioinformatic analyses were performed using Proteome Discoverer version 2.2 and Ingenuity Pathway Analysis. We identified a total of 1969 proteins, of which 1871 were quantified by TMT labels. Sixty-four proteins were expressed significantly ( p

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.

Sex differences in mitochondrial numbers and function are present in large cerebral arteries, but it is unclear whether these differences extend to the microcirculation. We performed an assessment of mitochondria-related proteins in cerebral microvessels (MVs) isolated from young, male and female, Sprague-Dawley rats. MVs composed of arterioles, capillaries, and venules were isolated from the cerebrum and used to perform a 3 vs. 3 quantitative, multiplexed proteomics experiment utilizing tandem mass tags (TMT), coupled with liquid chromatography/mass spectrometry (LC/MS). MS data and bioinformatic analyses were performed using Proteome Discoverer version 2.2 and Ingenuity Pathway Analysis. We identified a total of 1,969 proteins, of which 1,871 were quantified by TMT labels. Sixty-four proteins were expressed significantly (p < 0.05) higher in female samples compared with male samples. Females expressed more mitochondrial proteins involved in energy production, mitochondrial membrane structure, anti-oxidant enzyme proteins, and those involved in fatty acid oxidation. Conversely, males had higher expression levels of mitochondria-destructive proteins. We validated our key Proteomics results with western blotting. Our findings reveal, for the first time, the full extent of sexual dimorphism in the mitochondrial metabolic protein profiles of MVs, which may contribute to sex-dependent cerebrovascular and neurological pathologies.SynopsisEnergy-producing proteins in the cerebral microvessels (MVs) of male and female rats were examined by quantitative discovery-based proteomics to gain insight into the sex-dependent etiology of cardiovascular and neurological diseases. Females expressed more mitochondrial proteins involved in energy production, membrane structure, anti-oxidant activity, and fatty acid oxidation. In contrast, males exhibited more mitochondria-destructive proteins such as mitochondrial eating protein. Our findings reveal for the first time the sexual dimorphism of mitochondria-related proteins in cerebral MVs, which may explain functional sex-related differences in MVs during health and in the etiology of neurological pathologies of cerebrovascular origin.

One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance ( P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS. NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016.

The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7–10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATPchannels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.

Little is known about mitochondrial functioning in the cerebral vasculature during insulin resistance (IR). We examined mitochondrial respiration in isolated cerebral arteries of male Zucker obese (ZO) rats and phenotypically normal Zucker lean (ZL) rats using the Seahorse XFe24 analyzer. We investigated mitochondrial morphology in cerebral blood vessels as well as mitochondrial and nonmitochondrial protein expression levels in cerebral arteries and microvessels. We also measured reactive oxygen species (ROS) levels in cerebral microvessels. Under basal conditions, the mitochondrial respiration components (nonmitochondrial respiration, basal respiration, ATP production, proton leak, and spare respiratory capacity) showed similar levels among the ZL and ZO groups with the exception of maximal respiration, which was higher in the ZO group. We examined the role of nitric oxide by measuring mitochondrial respiration following inhibition of nitric oxide synthase with Nω-nitro-l-arginine methyl ester (l-NAME) and mitochondrial activation after administration of diazoxide (DZ). Both ZL and ZO groups showed similar responses to these stimuli with minor variations. l-NAME significantly increased the proton leak, and DZ decreased nonmitochondrial respiration in the ZL group. Other components were not affected. Mitochondrial morphology and distribution within vascular smooth muscle and endothelium as well as mitochondrial protein levels were similar in the arteries and microvessels of both groups. Endothelial nitric oxide synthase (eNOS) and ROS levels were increased in cerebral microvessels of the ZO. Our study suggests that mitochondrial function is not significantly altered in the cerebral vasculature of young ZO rats, but increased ROS production might be due to increased eNOS in the cerebral microcirculation during IR.

Clinical and preclinical studies have suggested a link between cardiovascular disease and dementia disorders, but the role of the collateral brain circulation in cognitive dysfunction remains unknown. We aimed to test the hypothesis that leptomeningeal arteriole (LMA) function and response to metabolic stressors differ among subjects with dementia, mild cognitive impairment (MCI), and normal cognition (CN). After rapid autopsy, LMAs were isolated from subjects with CN (n = 10), MCI (n = 12), or dementia [n = 42, Alzheimer’s disease (AD), vascular dementia (VaD), or other dementia], and endothelial and smooth muscle-dependent function were measured at baseline and after exposure to β-amyloid (2 μM), palmitic acid (150 μM), or medin (5 μM) and compared. There were no differences among the groups in baseline endothelial function (maximum dilation to acetylcholine, CN: 74.1 ± 9.7%, MCI: 67.1 ± 4.8%, AD: 74.7 ± 2.8%, VaD: 72.0 ± 5.3%, and other dementia: 68.0 ± 8.0%) and smooth muscle-dependent function (CN: 93.4 ± 3.0%, MCI: 83.3 ± 4.1%, AD: 91.8 ± 1.7%, VaD: 91.7 ± 2.4%, and other dementia: 87.9 ± 4.9%). There was no correlation between last cognitive function score and baseline endothelial or smooth muscle-dependent function. LMA endothelial function and, to a lesser extent, smooth muscle-dependent function were impaired posttreatment with β-amyloid, palmitic acid, and medin. Posttreatment LMA responses were not different between subjects with CN/MCI vs. dementia. Baseline responses and impaired vasoreactivity after treatment with metabolic stressors did not differ among subjects with CN, MCI, and dementia. The results suggest that the cognitive dysfunction in dementia disorders is not attributable to differences in baseline brain collateral circulation function but may be influenced by exposure of the vasculature to metabolic stressors. NEW & NOTEWORTHY Here, we present novel findings that brain collateral arteriole function did not differ among subjects with normal cognition, mild cognitive impairment, and dementia (Alzheimer’s disease and vascular dementia). Although arteriole function was impaired by vascular stressors (β-amyloid, palmitic acid, and medin), responses did not differ between those with or without dementia. The cognitive dysfunction in dementia disorders is not attributable to differences in baseline brain collateral circulation function but may be influenced by vascular exposure to metabolic stressors.

Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using Nω-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I–V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury.

We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 μM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.

The underlying factors promoting increased mitochondrial proteins, mtDNA, and dilation to mitochondrial-specific agents in male rats following tMCAO are not fully elucidated. Our goal was to determine the morphological and functional effects of ischemia/reperfusion (I/R) on mitochondria using electron microscopy, Western blot, mitochondrial oxygen consumption rate (OCR), and Ca2+ sparks activity measurements in middle cerebral arteries (MCAs) from male Sprague Dawley rats (Naïve, tMCAO, Sham). We found a greatly increased OCR in ipsilateral MCAs (IPSI) compared with contralateral (CONTRA), Sham, and Naïve MCAs. Consistent with our earlier findings, the expression of Mitofusin-2 and OPA-1 was significantly decreased in IPSI arteries compared with Sham and Naïve. Mitochondrial morphology was disrupted in vascular smooth muscle, but morphology with normal and perhaps greater numbers of mitochondria were observed in IPSI compared with CONTRA MCAs. Consistently, there were significantly fewer baseline Ca2+ events in IPSI MCAs compared with CONTRA, Sham, and Naïve. Mitochondrial depolarization significantly increased Ca2+ sparks activity in the IPSI, Sham, Naïve, but not in the CONTRA group. Our data indicate that altered mitochondrial structure and function occur in MCAs exposed to I/R and that these changes impact not only OCR but Ca2+ sparks activity in both IPSI and CONTRA MCAs.

Objective: Mice with genetic deletion of endothelial (eNOS; protective) and neuronal (nNOS; detrimental) nitric oxide synthase isoforms exhibit dramatically opposite consequences of ischemic brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify nNOS and characterize its functional role in primary brain microvascular endothelial cells (MECs). Methods and Results: MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used. In addition, rat brain microvessels were freshly isolated. Transendothelial electrical resistance (TEER) measurements of monolayers of hMECs cultured in transwells were used to quantitate in vitro blood-brain barrier (BBB) integrity. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in MECs but stained negative for glial (GFAP) and neuronal (Neu1) markers. PCR studies confirmed the expression of eNOS and nNOS mRNA in MECs and microvessels. We utilized electron spin resonance spectrometry to measure reactive oxygen species (ROS) (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; CMH) and NO (colloid Fe(DETC)2). Inhibition of nNOS (N-ω-Propyl-L-arginine and ARL-17477) reduced ROS but increased NO levels in MECs and rat brain microvessels. In contrast, eNOS inhibitor (L-N5-(1-Iminoethyl)ornithine) increased ROS but reduced NO levels. Inhibition of nNOS in neurons, similarly increased ROS and decreased NO levels. siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA as well as ROS levels. BBB studies of hMECs treated with NOS inhibitors followed by oxygen-glucose deprivation (OGD) revealed that nNOS inhibition increased TEER at baseline and promoted TEER recovery following OGD. In contrast, eNOS inhibition had no effect on TEER at baseline but weakly albeit transiently helps in the post-OGD recovery of BBB function. Conclusions: Thus, we identified a constitutively active nNOS in MECs that is functionally distinct from the nNOS isoform expressed in neurons and eNOS. In addition, nNOS inhibition enhances the BBB integrity and affords protection against anoxic-injury induced impairment of BBB function.

Mitochondrial dysfunction has been suggested as a potential underlying cause of pathological conditions associated with type 2 diabetes (T2DM). We have previously shown that mitochondrial respiration and mitochondrial protein levels were similar in the large cerebral arteries of insulin-resistant Zucker obese rats and their lean controls. In this study, we extend our investigations into the mitochondrial dynamics of the cerebral vasculature of 14-week-old Zucker diabetic fatty obese (ZDFO) rats with early T2DM. Body weight and blood glucose levels were significantly higher in the ZDFO group, and basal mitochondrial respiration and proton leak were significantly decreased in the large cerebral arteries of the ZDFO rats compared with the lean controls (ZDFL). The expression of the mitochondrial proteins total manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC) were significantly lower in the cerebral microvessels, and acetylated MnSOD levels were significantly reduced in the large arteries of the ZDFO group. Additionally, superoxide production was significantly increased in the microvessels of the ZDFO group. Despite evidence of increased oxidative stress in ZDFO, exogenous SOD was not able to restore mitochondrial respiration in the ZDFO rats. Our results show, for the first time, that mitochondrial respiration and protein levels are compromised during the early stages of T2DM.

Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by generation of mitochondrial reactive oxygen species (ROS), which sequentially promotes frequency of calcium sparks and activation of large conductance calcium-activated potassium channels (BKCa) in vascular smooth muscle (VSM). We previously demonstrated that cerebrovascular insulin resistance accompanies aging and obesity. It is unclear whether mitochondrial depolarization without the ROS generation enhances calcium sparks and vasodilation in phenotypically normal [Sprague Dawley (SD); Zucker lean (ZL)] and insulin-resistant [Zucker obese (ZO)] rats. We compared the mechanisms underlying the vasodilation to ROS-dependent (diazoxide) and ROS-independent [BMS-191095 (BMS)] mitoKATP channel activators in normal and ZO rats. Arterial diameter studies from SD, ZL, and ZO rats showed that BMS as well as diazoxide induced vasodilation in endothelium-denuded cerebral arteries. In normal rats, BMS-induced vasodilation was mediated by mitochondrial depolarization and calcium sparks generation in VSM and was reduced by inhibition of BKCa channels. However, unlike diazoxide-induced vasodilation, scavenging of ROS had no effect on BMS-induced vasodilation. Electron spin resonance spectroscopy confirmed that diazoxide but not BMS promoted vascular ROS generation. BMS- as well as diazoxide-induced vasodilation, mitochondrial depolarization, and calcium spark generation were diminished in cerebral arteries from ZO rats. Thus pharmacological depolarization of VSM mitochondria by BMS promotes ROS-independent vasodilation via generation of calcium sparks and activation of BKCa channels. Diminished generation of calcium sparks and reduced vasodilation in ZO arteries in response to BMS and diazoxide provide new insights into mechanisms of cerebrovascular dysfunction in insulin resistance.

AIMS: Type 2 diabetes mellitus is frequently associated with hypertension, but the underlying mechanisms are not completely understood. We tested the hypothesis that activation of type 1 prostaglandin E(2) (PGE(2)) receptor (EP1) increases skeletal muscle arteriolar tone and blood pressure in mice with type 2 diabetes. METHODS AND RESULTS: In 12-week-old, male db/db mice (with homozygote mutation in leptin receptor), systolic blood pressure was significantly elevated, compared with control heterozygotes. Isolated, pressurized gracilis muscle arterioles ( approximately 90 microm) of db/db mice exhibited an enhanced pressure- and angiotensin II (0.1-10 nM)-induced tone, which was reduced by the selective EP1 receptor antagonist, AH6809 (10 microM), to the level observed in arterioles of control mice. Exogenous application of PGE(2) (10 pM-100 nM) or the selective agonist of the EP1 receptor, 17-phenyl-trinor-PGE(2) (10 pM-100 nM), elicited arteriolar constrictions that were significantly enhanced in db/db mice (max: 31 +/- 4 and 29 +/- 5%), compared with controls (max: 20 +/- 2 and 14 +/- 3%, respectively). In the aorta of db/db mice, an increased protein expression of EP1, but not EP4, receptor was also detected by western immunoblotting. Moreover, we found that oral administration of the EP1 receptor antagonist, AH6809 (10 mg/kg/day, for 4 days), significantly reduced the systolic blood pressure in db/db, but not in control mice. CONCLUSION: Activation of EP1 receptors increases arteriolar tone, which could contribute to the development of hypertension in the db/db mice.

Mitochondrial initiated events of the diverse cells types comprising the neurovascular unit promote changes in cerebrovascular tone through multiple signaling pathways. Activation of the adenosine triphosphate (ATP)-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels) leads to mitochondrial depolarization as well as activation of cell-specific signaling mechanisms in endothelium, vascular smooth muscle (VSM), and perivascular and parenchymal nerves resulting in an integrated dilator response of cerebral arteries. Activation of mitoKATP channels relaxes VSM via generation of calcium sparks and subsequent downstream signaling mechanisms, and this relaxation can be augmented by nitric oxide (NO) produced by mitoKATP channel activation in endothelium and adjacent neurons. Some research suggests that calcium activated potassium channels may also be present in mitochondria (mitoKCa channels) and may affect cerebral vascular tone, but more research is needed to support this view. Pre-existing chronic conditions such as insulin resistance (IR) and/or diabetes impair mitoKATP channel-relaxation of cerebral arteries. Surprisingly, mitoKATP channel function after intense stress such as ischemia appears to be retained in large cerebral arteries despite generalized cerebral vascular dysfunction. Production of vasoactive factors following activation of mitochondria in response to physiological stimuli in one or more of the cells comprising the neurovascular unit may represent the elusive signaling link between metabolic rate and blood flow. In addition, our data indicate that mitoKATP channels represent an important, but underutilized target toward improving vascular dysfunction and decreasing brain injury in stroke patients.

BACKGROUND AND PURPOSE The transient receptor potential vanilloid 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli and is a therapeutic target. However, functional TRPV1 that affect microvascular diameter are also expressed in peripheral arteries and we attempted to characterize this receptor. EXPERIMENTAL APPROACH Sensory TRPV1 activation was measured in rats by use of an eye wiping assay. Arteriolar TRPV1-mediated smooth muscle specific responses (arteriolar diameter, changes in intracellular Ca2+) were determined in isolated, pressurized skeletal muscle arterioles obtained from the rat and wild-type or TRPV1−/− mice and in canine isolated smooth muscle cells. The vascular pharmacology of the TRPV1 agonists (potency, efficacy, kinetics of action and receptor desensitization) was determined in rat isolated skeletal muscle arteries. KEY RESULTS Capsaicin evoked a constrictor response in isolated arteries similar to that mediated by noradrenaline, this was absent in arteries from TRPV1 knockout mice and competitively inhibited by TRPV1 antagonist AMG9810. Capsaicin increased intracellular Ca2+ in the arteriolar wall and in isolated smooth muscle cells. The TRPV1 agonists evoked similar vascular constrictions (MSK-195 and JYL-79) or were without effect (resiniferatoxin and JYL-273), although all increased the number of responses (sensory activation) in the eye wiping assay. Maximal doses of all agonists induced complete desensitization (tachyphylaxis) of arteriolar TRPV1 (with the exception of capsaicin). Responses to the partial agonist JYL-1511 suggested 10% TRPV1 activation is sufficient to evoke vascular tachyphylaxis without sensory activation. CONCLUSIONS AND IMPLICATIONS Arteriolar TRPV1 have different pharmacological properties from those located on sensory neurons in the rat.

Aims Doxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turn causes DNA strand breakage culminating in poly(ADP-ribose) polymerase (PARP) activation and mitochondrial and cellular dysfunction. DNA nicks can activate numerous enzymes, such as PARP-2. Depletion of PARP-2 has been shown to result in a protective phenotype against free radical-mediated diseases, suggesting similar properties in the case of DOX-induced vascular damage. Methods and results PARP-2+/+ and PARP-2−/− mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg or 3 μM, respectively). Aortas were harvested 2-day post-treatment while MOVAS cells were treated with DOX for 7 hours. Aortas from PARP-2−/− mice displayed partial protection against DOX toxicity, and the protection depended on the conservation of smooth muscle but not on the conservation of endothelial function. DOX treatment evoked free radical production, DNA breakage and PARP activation. Importantly, depletion of PARP-2 did not quench any of these phenomena, suggesting an alternative mechanism. Depletion of PARP-2 prevented DOX-induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP-2 resulted in the induction of the SIRT1 promoter and consequently increased SIRT1 expression both in aortas and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis, which provided protection against DOX-induced mitochondrial damage. Conclusion Our data identify PARP-2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to the best of our knowledge, this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress.