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As quoted by the famous astronomer Carl Sagan, “the absence of evidence is not the evidence of absence” (http://wiki.c2.com/?AbsenceOfEvidenceIsNotEvidenceOfAbsence) is the logical corollary that conclusions should not be drawn when a direct test for the presence of evidence cannot be made. This applies to the article by Matrone et al1 in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , where the authors delineated a novel signaling pathway within the domain of endothelial migration. LMO2 (LIM domain only 2) was first discovered as a recurrent translocation partner of T-cell receptor loci in a subset of patients with T-cell acute lymphoblastic leukemia in 1981.2,3 Studies on LMO2 have now shown that LMO2 functions as a proto-oncogenic gene not only in T cells but also in hematopoiesis, vascular remodeling, and stem cell biology.4–9 Matrone et al1 showed that LMO2 levels in zebrafish embryos regulate intersegmental vessel (ISV) formation. The evidence for a migration defect is the absence of endothelial cells in ISVs in the elegant photomicrographs. Of course direct visualization of the lack of endothelial migration cannot be presented. See accompanying article on page 1860 In transgenic zebrafish that express enhanced GFP (green fluorescent protein; under the control of Fli1 [friend leukemia integration 1 transcription factor]) in the vasculature (Tg(fli1:EGFP)y1),10 the authors show that total proliferating endothelial cells were not different in the LMO2 inhibited embryos versus controls, but GFP+ endothelial-like cells accumulate in the larger conduits with lesser numbers in connecting ISVs allowing the conclusion of impaired migration into the ISVs. Using a stable human umbilical vein endothelial cell (HUVEC) line deficient …

This editorial refers to ‘Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase’ by S. Werfel et al. , pp. 15–23, this issue. Knockout mice have revolutionized our ability to conduct biomedical research and no area has benefited more than cardiovascular disease. The knockout (or genetic deletion) of (a) specific gene(s) has provided a clearer understanding of the roles a gene plays in development, normal physiology, and in responses to injury, vasomotor regulation, and excitation/contraction coupling to name a few. These advances notwithstanding, the utility of traditional knockout mice can be limited in cases where the gene deletion causes embryonic lethality, or when a tissue-specific knockout is desired to avoid having off-target effects limit the study. The application of Cre-lox technology to transgenesis has provided a powerful tool in helping investigators create tissue-specific knockouts.1 The Cre-lox strategy requires the breeding of two separate lines of genetically manipulated mice: ‘floxed’ mice in which the gene of interest has been flanked by two loxP sites using ‘knock-in’ techniques, and another line of transgenic mice in which a tissue-specific promoter is used to drive the expression of Cre-recombinase. By crossing these two strains, mice are obtained in which tissue-specific expression of Cre results in deletion of the gene of interest due to the action of the Cre-recombinase enzyme that excises the genomic region flanked by the loxP sites. …

Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice,We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia.We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment ofTaken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Myoendothelial junctions are specialised projections of cell : cell contact through the internal elastic lamina between endothelial cells and vascular smooth muscle cells. These junctions allow for endothelial cells and vascular smooth muscle cells to make direct membrane apposition and are involved in cell : cell communication. In this study, we evaluated for the presence of myoendothelial junctions in murine corporal tissue and used plasminogen activator inhibitor (PAI)-1-deficient mice, which lack myoendothelial junctions, to determine whether myoendothelial junctions affect erectile function. Transmission electron microscopy demonstrated the presence of myoendothelial junctions in the corporal tissue of wild-type mice and confirmed the decreased junction numbers in the tissue of PAI-1(-/-) mice. A potential role for myoendothelial junctions in tumescence was established; in that, PAI-1(-/-) mice demonstrated a significantly longer time to achieve maximal intracavernous pressure. Treatment of PAI-1(-/-) mice with recombinant PAI-1 restored the number of myoendothelial junctions in the corporal tissue and also induced a significant decrease in time to maximal corporal pressures. Myoendothelial junctions were similarly identified in the human corporal tissue. These results suggest a critical role for myoendothelial junctions in erectile pathophysiology and therapies aimed at restoring myoendothelial junction numbers in the corporal tissue may provide a novel therapy for erectile dysfunction.

Purpose Gene-modified mice may be used to elucidate molecular mechanisms underlying abnormal myocardial blow flow (MBF). We sought to develop a quantitative myocardial perfusion imaging technique for mice and to test the hypothesis that myocardial perfusion reserve (MPR) is reduced in a mouse model of diet-induced obesity (DIO). Methods A dual-contrast saturation-recovery sequence with ky-t undersampling and a motion-compensated compressed sensing reconstruction algorithm was developed for first-pass MRI on a small-bore 7 Tesla system. Control mice were imaged at rest and with the vasodilators ATL313 and Regadenoson (n = 6 each). In addition, we imaged mice fed a high-fat diet (HFD) for 24 weeks. Results In control mice, MBF was 5.7 ± 0.8 mL/g/min at rest and it increased to 11.8 ± 0.6 mL/g/min with ATL313 and to 10.4 ± 0.3 mL/g/min with Regadenoson. In HFD mice, we detected normal resting MBF (5.6 ± 0.4 versus 5.0 ± 0.3 on control diet), low MBF at stress (7.7 ± 0.4 versus 10.4 ± 0.3 on control diet, P

Macrophages respond to signals in the microenvironment by changing their functional phenotypes, a process known as polarization. Depending on the context, they acquire different patterns of transcriptional activation, cytokine expression and cellular metabolism which collectively constitute a continuous spectrum of phenotypes, of which the two extremes are denoted as classical (M1) and alternative (M2) activation. To quantitatively decode the underlying principles governing macrophage phenotypic polarization and thereby harness its therapeutic potential in human diseases, a systems-level approach is needed given the multitude of signaling pathways and intracellular regulation involved. Here we develop the first mechanism-based, multi-pathway computational model that describes the integrated signal transduction and macrophage programming under M1 (IFN-γ), M2 (IL-4) and cell stress (hypoxia) stimulation. Our model was calibrated extensively against experimental data, and we mechanistically elucidated several signature feedbacks behind the M1-M2 antagonism and investigated the dynamical shaping of macrophage phenotypes within the M1-M2 spectrum. Model sensitivity analysis also revealed key molecular nodes and interactions as targets with potential therapeutic values for the pathophysiology of peripheral arterial disease and cancer. Through simulations that dynamically capture the signal integration and phenotypic marker expression in the differential macrophage polarization responses, our model provides an important computational basis toward a more quantitative and network-centric understanding of the complex physiology and versatile functions of macrophages in human diseases., Author summary As essential regulators of the immune system, macrophages can be polarized to acquire distinct phenotypes in response to a wide range of signals in the tissue microenvironment, such as bacterial products, endogenous cytokines, cell damage and stress. Decades of research has shown that a number of signaling pathways can regulate this process and determine the functional phenotypes of macrophages in physiology as well as various disease scenarios, and recent studies suggest that macrophage polarization is indeed a dynamic process and that the canonical dichotomous notion with only classical (M1) and alternative (M2) activation states is oversimplifying the continuous spectrum of polarized macrophage phenotypes observed in health and disease. To investigate the mechanistic and therapeutic aspects associated with differentially polarized macrophages, we formulated and calibrated a multi-pathway computational model based on literature knowledge and quantitative experimental datasets to systematically describe the integrative regulation of macrophage transcriptional programs and phenotype markers under different stimuli combinations. Our systems-level model is a key building block of a potential “virtual macrophage” simulation platform that can enable researchers to efficiently generate mechanistic hypotheses and assess macrophage-based therapeutic strategies for human diseases.

Background In patients with peripheral artery disease (PAD), the severity of symptoms correlates poorly with ankle-brachial index (ABI). The aim of this study was to test the hypothesis that limb perfusion assessed using contrast-enhanced ultrasound (CEU) during contractile exercise varies according to functional class in patients with PAD, particularly those with ABIs in the 0.4 to 0.6 range whose symptoms vary widely. Methods Bilateral quantitative CEU perfusion imaging of the calf was performed in normal control subjects (n = 10) and patients with PAD who had at least one limb with a moderately reduced ABI (0.4–0.6; n = 17). Imaging was performed at rest and immediately after 30 sec of modest periodic (0.3-Hz) plantar flexion (10 W). Results In patients with PAD, Rutherford symptom classification for each limb varied widely, including in limbs with ABIs of 0.4 to 0.6 (n = 6 with mild or no symptoms, n = 14 with moderate to severe symptoms). CEU perfusion imaging parameters at rest were similar between control subjects and patients with PAD irrespective of ABI. In normal control subjects, limb flow increased on average by > 20-fold after only 30 sec of moderate exercise. In patients with PAD, muscle exercise perfusion for all limbs was reduced compared with control subjects and decreased according to the severity of ABI reduction, primarily from reduced microvascular flux rate. Even limbs with ABIs > 0.9 in patients with PAD had lower exercise perfusion than in control subjects (P = .03). In subjects with PAD, exercise perfusion was lower in those with moderate to severe versus mild symptoms when analyzed for all limbs (median, 30 IU/sec [interquartile range (IQR), 21–52 IU/sec] vs 84 IU/sec [IQR, 36–177 IU/sec]; P = .01) and limbs with ABIs of 0.4 to 0.6 (median, 26 IU/sec [IQR, 14–41 IU/sec] vs 54 IU/sec [IQR, 31–105 IU/sec]; P = .05). Conclusions In patients with PAD, CEU exercise perfusion imaging detects differences in limb muscle perfusion that are likely to be responsible for differences in symptom severity and can detect the flow abnormalities from microvascular dysfunction even in limbs with normal ABIs.

The current (1972) status of cable television (CATV) in Massachusetts is reviewed in this guide. The tone of the guide is oriented toward more citizen participation and public access to CATV operations throughout the state. The legal and technical CATV considerations are briefly discussed. Short illustrated chapters on the possibilities of local programing, community involvement, education, and franchising are presented. Of particular importance are the tables, maps, and listings arranged throughout the guide that reveal the ownership of the CATV operations, the areas with cable, and market information. The guide is intended for any person or organization interested in cable television. (MC)

Aims To determine if type 2 diabetes mellitus (T2D) differentiates endothelial function and plasma nitrite response (a marker of nitric oxide bioavailability) during exercise in peripheral arterial disease (PAD) subjects prior to and following 3 months supervised exercise training (SET). Methods In subjects with T2D + PAD (n = 13) and PAD-only (n = 14), endothelial function was measured using brachial artery flow-mediated dilation. On a separate day, venous blood draws were performed at rest and 10 min following a symptom-limited graded treadmill test (SL-GXT). Plasma samples were snap-frozen for analysis of nitrite by reductive chemiluminescence. All testing was repeated following 3 months of SET. Results Prior to training both groups demonstrated endothelial dysfunction, which was correlated with a net decrease in plasma nitrite following a SL-GXT (p ≤ 0.05). Following SET, the PAD-only group demonstrated an improvement in endothelial function (p ≤ 0.05) and COT (p ≤ 0.05), which was related to a net increase in plasma nitrite following the SL-GXT (both p ≤ 0.05). The T2D + PAD group had none of these increases. Conclusions T2D in the presence of PAD attenuated improvements in endothelial function, net plasma nitrite, and COT following SET. This suggests that T2D maybe associated with an inability to endogenously increase vascular NO bioavailability to SET.

Aims Diabetes mellitus (DM) is associated with poor clinical outcomes in humans with peripheral arterial disease (PAD) and in pre-clinical models of PAD, but the effects of glycaemic control are poorly understood. We investigated the effect of glycaemic control on experimental PAD in mice with Type 1 DM and explored the effects of hyperglycaemia on vascular endothelial growth factor receptor 2 (VEGFR2) expression in ischaemia. Methods and results Hind limb ischaemia was induced in non-diabetic, untreated Type 1 DM, and treated Type 1 DM mice. We assessed perfusion recovery, capillary density, VEGFR2 levels, and VEGFR2 ubiquitination in ischaemic hind limbs. We found that untreated Type 1 DM mice showed impaired perfusion recovery, lower hind limb capillary density 5 weeks post-ischaemia, and lower VEGFR2 protein in Day 3 post-ischaemic hind limbs when compared with non-DM controls. Treated Type 1 DM mice had perfusion recovery, capillary density, and VEGFR2 protein levels comparable with that of non-diabetic mice at the same time points. Treatment with anti-VEGFR2 antibody negated that the improved perfusion recovery displayed by treated Type 1 DM mice. In ischaemic Type 1 DM hind limbs and endothelial cells exposed to simulated ischaemia, high glucose impaired VEGFR2 expression and was associated with increased VEGFR2 ubiquitination. Inhibition of the ubiquitin–proteasome complex restored normal endothelial VEGFR2 expression in simulated ischaemia. Conclusion Hyperglycaemia in Type 1 DM impairs VEGFR2 protein expression in ischaemic hind limbs, likely due to increased ubiquitination and degradation by the proteasome complex. Glycaemic control allows normal levels of VEGFR2 in ischaemia and improved perfusion recovery.

Nitrite stores decrease after exercise in patients with peripheral artery disease (PAD) and diabetes represents decreased nitric oxide (NO) bioavailability that may contribute to endothelial dysfunction and limit exercise duration. The primary objective of this placebo-controlled study was the safety and tolerability of multiple doses of oral sodium nitrite in patients with PAD, predominantly with diabetes, over a period of 10 weeks. The primary efficacy endpoint was endothelial flow-mediated dilatation (FMD) and secondary efficacy endpoints included a 6-minute walk test and quality of life assessment. Of the 55 subjects, the most common side effects attributed to sodium nitrite were a composite of headache and dizziness occurring in 21% with the 40 mg dose and 44% with the 80 mg dose. There was no clinically significant elevation of methemoglobin. FMD non-significantly worsened in the placebo and 40 mg groups, but was stable in the 80 mg group. Diabetic patients receiving 80 mg had significantly higher FMD compared with the placebo and 40 mg groups. There was no significant change in 6-minute walk test or quality of life parameters over time compared to placebo. In conclusion, sodium nitrite therapy is well tolerated in patients with PAD. The possible clinical benefit of sodium nitrite should be studied in a larger and fully powered trial.

Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg(+)) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg(+) mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg(+) mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease.

Functional impairment, functional decline, and mobility loss are major public health problems in people with lower extremity peripheral artery disease (PAD). Few medical therapies significantly improve walking performance in PAD. We describe methods for the PROgenitor cell release Plus Exercise to improve functionaL performance in PAD (PROPEL) Study, a randomized controlled clinical trial designed to determine whether granulocyte-macrophage colony stimulating factor (GM-CSF) combined with supervised treadmill walking exercise improves six-minute walk distance more than GM-CSF alone, more than supervised treadmill exercise alone, and more than placebo plus attention control in participants with PAD, respectively. PROPEL Study participants are randomized to one of four arms in a 2 by 2 factorial design. The four study arms are GM-CSF plus supervised treadmill exercise, GM-CSF plus attention control, placebo plus supervised exercise therapy, or placebo plus attention control. The primary outcome is change in six-minute walk distance at 12-week follow-up. Secondary outcomes include change in brachial artery flow-mediated dilation (FMD), change in maximal treadmill walking time, and change in circulating CD34+ cells at 12-week follow-up. Outcomes are also measured at six-week and six-month follow-up. Results of the PROPEL Study will have important implications for understanding mechanisms of improving walking performance and preventing mobility loss in the large and growing number of men and women with PAD.

Background— MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia. Methods and Results— From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types. Conclusions— Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

VEGF receptor (VEGFR) cell surface localization plays a critical role in transducing VEGF signaling toward angiogenic outcomes, and quantitative characterization of these parameters is critical to advancing computational models for predictive medicine. However, studies to this point have largely examined intact muscle; thus, essential data on the cellular localization of the receptors within the tissue are currently unknown. Therefore, our aims were to quantitatively analyze VEGFR localization on endothelial cells (ECs) from mouse hindlimb skeletal muscles after the induction of hindlimb ischemia, an established model for human peripheral artery disease. Flow cytometry was used to measure and compare the ex vivo surface localization of VEGFR1 and VEGFR2 on CD31+/CD34+ECs 3 and 10 days after unilateral ligation of the femoral artery. We determined that 3 days after hindlimb ischemia, VEGFR2 surface levels were decreased by 80% compared with ECs from the nonischemic limb; 10 days after ischemia, we observed a twofold increase in surface levels of the modulatory receptor, VEGFR1, along with increased proliferating cell nuclear antigen, urokinase plasminogen activator, and urokinase plasminogen activator receptor mRNA expression compared with the nonischemic limb. The significant upregulation of VEGFR1 surface levels indicates that VEGFR1 indeed plays a critical role in the ischemia-induced perfusion recovery process, a process that includes both angiogenesis and arteriogenesis. The quantification of these dissimilarities, for the first time ex vivo, provides insights into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors in ischemia and lays the foundation for systems biology approaches toward therapeutic angiogenesis.

Atherosclerosis is the leading cause of morbidity and mortality in the Western world. Peripheral artery disease (PAD) has been less studied then coronary artery disease but is nearly as common. PAD impairs blood flow to the leg(s) and causes functional impairment, leg pain, and amputation. The last drug approved for PAD was in 1999. Blood flow to leg proceeds through one major artery and in PAD total occlusions in the course of that vessel are common. Thus, the extent of new blood vessel growth determines a patients’ clinical course. Promoting the growth of new blood vessels (therapeutic angiogenesis) was a major goal of therapy. Results from studies using cytokine growth factors have shown disappointing results. Using clinical and preclinical studies, our laboratory has identified several novel therapeutic approaches. One, a modulator of innate immunity, will be reviewed as an approach that has the potential to create new therapies for PAD.

The world of construction is subject to growing requirements regarding the quality of buildings. The delivery of an envelope must be accompanied by an EPB certificate. In addition, contractors are requiring levels of performance that are higher than those imposed by the regulation: BREEAM, LEED or passive certifications. These certifications are based on theoretical calculations and do not provide a guarantee of real building performance. However contractors aspire to obtain better guarantees regarding buildings real performance. The increase in the number of airtightness tests performed reflects this concern. In 2014, one out of every three new dwellings is subject to air-tightness measures in the Flemish Region and one in five in the Walloon Region of Belgium. In that context, there is a growing interest in the concept of ‘energy performance contracting’ defined by the European Directive 2012/27/EU as “a contractual arrangement between the beneficiary and the provider of an energy efficiency improvement measure, verified and monitored during the whole term of the contract, where investments (work, supply or service) in that measure are paid for in relation to a contractually agreed level of energy efficiency improvement or other agreed energy performance criterion, such as financial savings”. Being able to confirm that the obligation of result is achievable and achieved, is the new challenge faced by the construction industry. Research institutes want to consolidate sets of achievable reference values for energy performance indicators and to compare them with theoretical predicted values. They also intend to extend their expertise regarding the analysis of data collected by in situ testing. Education institutions that forms architect engineers intend to promote their training in the processing of data collected through building monitoring, as a significant amount of those engineers are involved in the management of building stocks, thereby confronted to large samples of measured data. Universities are also committed to serve the community. By promoting the quality of workmanship in the construction sector as well as the quality of the building stock, a guarantee of quality is provided to the consumers. New job opportunities could also be created in the measurement of building performances and the analysis of monitored data. In that context, Cambbridge research project is gathering a Belgian construction company (Jacques Delens s.a.) and a university (UCL) in order to develop reliable methods that can be applied on site during the construction phase in order to assess the actual performance of buildings at a moment when it can still be improved to reach the target without entailing over costs. The proposed measurement process includes 5 days of pre monitoring during which the building can be occupied or not, followed by 9 days monitoring without occupancy. It is a tradeoff between the constraints of the construction work, that do not allow too much time to be spend for the tests, and constraints related to building physics. The research implies parallel experiments on identical building units, to check the reliability of a method based the observation of floating indoor temperature compared to a conventional co heating test. The relevancy of the placement of internal insulation on party walls is also checked as it allows the indoor temperature to reach higher values and as it reduces heat losses to neighboring units. As quasi-stationary co-heating has so far been limited to the winter months, it is crucial to expand the time window for HLC assessment by developing summer approaches. Methods based on the observation of floating indoor temperature meet that goal. Such innovative measurements methods try to take advantage of solar gains effects instead of avoiding them.

Peripheral Arterial Disease (PAD) is one of the major complications of systemic atherosclerosis where occlusions along the major arterial pathway that supplies blood to the lower extremities is interrupted and blood flow to the distal limb becomes dependent on the presence, extent, and function of collateral blood vessels. Estimates are PAD is present in ~8.5 million Americans at or over the age of 40 and the two major clinical manifestations of PAD are intermittent claudication (IC) and critical limb ischemia (CLI) (Go et al., Circulation 129(3):e28–e292, 2014). Across the two major clinical manifestations of PAD the types of leg symptoms, amputation rates, and mortality differ greatly (Norgren et al., J Vasc Surg 45(Suppl S):S5–S67, 2007). Medical therapies for PAD subjects are designed to limit complications from systemic but no medical therapies are reliably able to improve blood flow to the ischemic limb. Here we will review how trials of therapeutic angiogenesis using gene or cell therapy have fared to treat PAD.

Angiogenesis is the growth of new blood vessels from pre-existing microvessels. Peripheral arterial disease (PAD) is caused by atherosclerosis that results in ischemia mostly in the lower extremities. Clinical trials including VEGF-A administration for therapeutic angiogenesis have not been successful. The existence of anti-angiogenic isoform (VEGF165b) in PAD muscle tissues is a potential cause for the failure of therapeutic angiogenesis. Experimental measurements show that in PAD human muscle biopsies the VEGF165b isoform is at least as abundant if not greater than the VEGF165a isoform. We constructed three-compartment models describing VEGF isoforms and receptors, in human and mouse, to make predictions on the secretion rate of VEGF165b and the distribution of various isoforms throughout the body based on the experimental data. The computational results are consistent with the data showing that in PAD calf muscles secrete mostly VEGF165b over total VEGF. In the PAD calf compartment of human and mouse models, most VEGF165a and VEGF165b are bound to the extracellular matrix. VEGF receptors VEGFR1, VEGFR2 and Neuropilin-1 (NRP1) are mostly in ‘Free State’. This study provides a computational model of VEGF165b in PAD supported by experimental measurements of VEGF165b in human and mouse, which gives insight of VEGF165b in therapeutic angiogenesis and VEGF distribution in human and mouse PAD model.

Peripheral arterial disease (PAD) is caused by atherosclerosis that results in narrowing and frequently complete occlusions of one or more arteries that supply the lower extremities. PAD affects ~8.5 million Americans at or over the age of 40 (Go et al., Circulation 129:e28–e292, 2014). The two major clinical manifestations of PAD are intermittent claudication (IC) and critical limb ischemia (CLI). Intermittent claudication is defined by the presence of leg pain/cramping with walking that relieves with rest. CLI is defined as pain present at rest, with or without ischemic ulcers or gangrene, classified as Rutherford-Becker Class 4–6 or Fontaine Class III and IV. Patients with CLI are quite different than those with IC. The 1-year mortality rate in patients with CLI is approximately 25 %, and the overall amputation rate over 1 year is approximately 30 % (Norgren et al., J Vasc Surg 45:S5–67, 2007). While medical therapies to limit complications from atherosclerosis reduce general cardiovascular mortality in patients with CLI, at present there is no definitive medical therapy for CLI. Newer medications such as Praxilene (a metabolic enhancer and a 5-HT2 receptor antagonist) and Alprostadil (a prostaglandin E1 analogue) have been recently approved for use in Europe in patients with PAD to improve blood flow to the ischemic limb. However, mixed results from the clinic studies indicate that these drugs need further long-term evaluation to establish their role in PAD.

Background: Currently, no therapies exist for treating and improving outcomes in patients with severe peripheral artery disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and to reduce tissue loss in genetic PAD models. However, the cell-specific function, downstream mechanisms, or signaling involved in miR93-mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD, and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1 activation/polarization state. In the present study, we identified a novel mechanism by which miR93 regulates macrophage polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental PAD. Methods: In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation conditions) and in vivo experiments in preclinical PAD models (unilateral femoral artery ligation and resection) were conducted to examine the role of miR93-interferon regulatory factor-9–immunoresponsive gene-1 (IRG1)–itaconic acid pathway in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery. Results: In vivo, compared with wild-type controls, miR106b-93-25 cluster–deficient mice (miR106b-93-25 −/− ) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like macrophages after experimental PAD. Intramuscular delivery of miR93 in miR106b-93-25 −/− PAD mice increased angiogenesis, arteriogenesis, and the extent of perfusion, which correlated with more M2-like macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like polarization even under M1-like polarizing conditions (hypoxia serum starvation). Delivery of bone marrow–derived macrophages from miR106b-93-25 −/− to wild-type ischemic muscle decreased angiogenesis, arteriogenesis, and perfusion, whereas transfer of wild-type macrophages to miR106b-93-25 −/− had the opposite effect. Systematic analysis of top differentially upregulated genes from RNA sequencing between miR106b-93-25 −/− and wild-type ischemic muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage polarization. The 3′ untranslated region luciferase assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not an miR93 target but that interferon regulatory factor-9, which can regulate IRG1 expression, is an miR93 target. In vitro, increased expression of interferon regulatory factor-9 and IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. Conclusions: miR93 inhibits interferon regulatory factor-9 to decrease IRG1–itaconic acid production to induce M2-like polarization in ischemic muscle to enhance angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.

Rationale: Atherosclerotic-arterial occlusions decrease tissue perfusion causing ischemia to lower limbs in patients with peripheral arterial disease (PAD). Ischemia in muscle induces an angiogenic response, but the magnitude of this response is frequently inadequate to meet tissue perfusion requirements. Alternate splicing in the exon-8 of vascular endothelial growth factor (VEGF)-A results in production of proangiogenic VEGF xxx a isoforms (VEGF 165 a, 165 for the 165 amino acid product) and antiangiogenic VEGF xxx b (VEGF 165 b) isoforms. Objective: The antiangiogenic VEGF xxx b isoforms are thought to antagonize VEGF xxx a isoforms and decrease activation of VEGF receptor-2 (VEGFR2), hereunto considered the dominant receptor in postnatal angiogenesis in PAD. Our data will show that VEGF 165 b inhibits VEGFR1 signal transducer and activator of transcription (STAT)-3 signaling to decrease angiogenesis in human and experimental PAD. Methods and Results: In human PAD versus control muscle biopsies, VEGF 165 b: (1) is elevated, (2) is bound higher (versus VEGF 165 a) to VEGFR1 not VEGFR2, and (3) levels correlated with decreased VEGFR1, not VEGFR2, activation. In experimental PAD, delivery of an isoform-specific monoclonal antibody to VEGF 165 b versus control antibody enhanced perfusion in animal model of severe PAD (Balb/c strain) without activating VEGFR2 signaling but with increased VEGFR1 activation. Receptor pull-down experiments demonstrate that VEGF 165 b inhibition versus control increased VEGFR1–STAT3 binding and STAT3 activation, independent of Janus-activated kinase-1)/Janus-activated kinase-2. Using VEGFR1 +/− mice that could not increase VEGFR1 after ischemia, we confirm that VEGF 165 b decreases VEGFR1–STAT3 signaling to decrease perfusion. Conclusions: Our results indicate that VEGF 165 b prevents activation of VEGFR1–STAT3 signaling by VEGF 165 a and hence inhibits angiogenesis and perfusion recovery in PAD muscle.

Proton pump inhibitors (PPIs) are a widely used category of drug for past 20 years. Originally requiring a prescription, PPIs were used to treat peptic ulcer and gastroesophageal reflux disease. PPIs now are readily available over the counter, and their use is often unmonitored. Over utilization of PPIs for inappropriate indications and their use for durations beyond that indicated in original recommendations have been reported in both hospital and ambulatory settings.1,2 Over the past few years, the safety of long-term PPI use has come into question with observational studies reporting increased prevalence of dementia, myocardial infarction, and renal failure in people who use long-term PPIs.3–6 However, no causative mechanism for the adverse effects of PPI has been established. Article, see p e36 Following their recent report of association of PPI use with myocardial infarction in the general population,5 in this issue of Circulation Research , Yepuri et al7 used a series of invitro studies using human microvascular endothelial cells (EC) treated with clinically relevant concentrations of the PPI, esomeprazole. They showed reduced lysosomal cathepsin-B and phosphatase, and increased lysosomal protein aggregates in ECs treated with esomeprazole versus controls, indicating impaired lysosomal proteostasis. Treatment with esomeprazole increased production of EC superoxide anions, decreased eNOS (endothelial nitric oxide synthase) and iNOS (inducible nitric oxide synthase) expression, and reduced nitric oxide (NO) production. In matrigel assays, treatment with esomeprazole reduced microvascular EC proliferation and network formation. Finally, the authors show that esomeprazole treatment accelerated EC senescence as …

Background Impaired myocardial perfusion reserve (MPR) is prevalent in obesity and diabetes, even in the absence of obstructive coronary artery disease (CAD), and is prognostic of adverse events. We sought to establish the time course of reduced MPR and to investigate associated vascular and tissue properties in mice fed a high-fat diet (HFD), as they are an emerging model of human obesity, diabetes, and reduced MPR without obstructive CAD. Methods C57Bl/6 mice fed a HFD or a low-fat diet (control) were imaged at 6, 12, 18 and 24 weeks post-diet. The cardiovascular magnetic resonance (CMR) protocol included multi-slice cine imaging to assess ejection fraction (EF), left-ventricular (LV) mass, LV wall thickness (LVWT), and LV volumes, and first-pass perfusion CMR to quantify MPR. Coronary vascular reactivity, aortic atherosclerosis, myocardial capillary density and tissue fibrosis were also assessed. Results Body weight was increased in HFD mice at 6–24 weeks post-diet (p

Ischemic vascular diseases such as coronary, carotid, and peripheral artery disease (PAD) require vascular growth and remodeling in order to restore blood flow to ischemic tissue. Human therapeutic studies aimed at promoting neovascularization have been largely unsuccessful in part because these approaches have focused on stimulating endothelial cell (EC) growth without coordinate investment of smooth muscle cells and pericytes (SMC-P) needed for functional blood vessels that are stable and able to regulate perfusion. Thus, there is a critical need to identify mechanisms that mediate perivascular cell coverage during vascular network remodeling, a process that requires perivascular cell detachment, migration, and investment of endothelial tubes. Previous work from the Owens lab demonstrated that the stem cell pluripotency gene Oct4, previously thought to be permanently epigenetically silenced in all somatic cells, is reactivated in SMC within atherosclerotic lesions. SMC specific KO of Oct4 results in marked reductions in the number of SMC within lesions due to failure of cells to migrate out of the media. That is, Oct4 reactivation appears to be critical for SMC migration, at least in the context of atherosclerosis. Therefore, the present study tested the hypothesis that SMC-P reactivation of Oct4 is essential for neovascularization following hindlimb ischemia (HLI), a mouse model of PAD. To test this hypothesis, we used a unique mouse line enabling SMC-P specific tamoxifen-inducible lineage tracing (termed Myh11 eYFP), allowing their tracking following phenotypic switching and loss of identifying genes, combined with SMC-P specific knockout of Oct4. We subjected both Oct4WT and Oct4KO Myh11 eYFP mice to HLI and monitored perfusion recovery via Laser Doppler at days 0, 3, 7, 14, and 21 post-HLI. We found that SMC-P specific KO of Oct4 results in significantly impaired perfusion recovery at days 14 and 21 post-HLI. We harvested hindlimb muscle at day 21 post-HLI and observed that Oct4 KO mice have significantly impaired angiogenesis, but intact arteriogenesis, compared to WT controls. Future studies will investigate Oct4 downstream targets necessary for angiogenesis following HLI.

Background Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2–3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. Methods AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. Results Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. Conclusions Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling. Copyright © 2012 John Wiley & Sons, Ltd.