Functional Vasodilation Research Articles

Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal α-Adrenergic Signaling and a Transient Loss of Vascular Tone.

Stimulating collateral arteriogenesis is an attractive therapeutic target for peripheral artery disease (PAD). However, the potency of arteriogenesis-stimulation in animal models has not been matched with efficacy in clinical trials. This may be because the presence of enlarged collaterals is not sufficient to relieve symptoms of PAD, suggesting that collateral function is also important. Specifically, collaterals are the primary site of vascular resistance following arterial occlusion, and impaired collateral vasodilation could impact downstream tissue perfusion and limb function. Therefore, we evaluated the effects of arteriogenesis on collateral vascular reactivity. Following femoral artery ligation in the mouse hindlimb, collateral functional vasodilation was impaired at day 7 (17 ± 3 vs. 60 ± 8%) but restored by day 28. This impairment was due to a high resting diameter (73 ± 4 μm at rest vs. 84 ± 3 μm dilated), which does not appear to be a beneficial effect of arteriogenesis because increasing tissue metabolic demand through voluntary exercise decreased resting diameter and restored vascular reactivity at day 7. The high diameter in sedentary animals was not due to sustained NO-dependent vasodilation or defective myogenic constriction, as there were no differences between the enlarged and native collaterals in response to eNOS inhibition with L-NAME or L-type calcium channel inhibition with nifedipine, respectively. Surprisingly, in the context of reduced vascular tone, vasoconstriction in response to the α-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (−62 ± 2 vs. −37 ± 2%) while vasodilation in response to the α-adrenergic antagonist prazosin was reduced (6 ± 4% vs. 22 ± 16%), indicating a lack of α-adrenergic receptor activation by endogenous norepinephrine and suggesting a denervation of the neuroeffector junction. Staining for tyrosine hydroxylase demonstrated sympathetic denervation, with neurons occupying less area and located further from the enlarged collateral at day 7. Inversely, MMP2 presence surrounding the enlarged collateral was greater at day 7, suggesting that denervation may be related to extracellular matrix degradation during arteriogenesis. Further investigation on vascular wall maturation and the functionality of enlarged collaterals holds promise for identifying novel therapeutic targets to enhance arteriogenesis in patients with PAD.

Reduced Flow-Mediated Dilatation Is Not Related to COVID-19 Severity Three Months after Hospitalization for SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has spread rapidly worldwide, with more than two million deaths. Evidence indicates the critical role of the vascular endothelium in its pathophysiology but, like potential changes in functional vasodilation, the vascular effect of SARS-CoV-2 at a given distance from the acute infection is largely unknown. We assessed brachial artery flow-mediated dilatation (FMD) in 27 COVID-19 patients needing conventional or intensive care unit hospitalization, three months after SARS-CoV-2 infection diagnosis and in nine age- and sex- matched control subjects. Interestingly, the FMD was lower in COVID-19 patients as compared to controls (8.2 (7.2–8.9) vs. 10.3 (9.1–11.7)); p = 0.002, and half of the hospitalized COVID-19 survivors presented with a reduced FMD < 8% at three months of COVID-19 onset. Impaired FMD was not associated with severe or critical SARS-CoV-2 infection, reflected by ICU hospitalization, total hospitalization duration, or severity of lung damage. In conclusion, reduced FMD is often observed even three months after hospitalization for SARS-CoV-2 infection, but such alteration predominantly appears to not be related to COVID-19 severity. Longer and larger follow-up studies will help to clarify the potential prognosis value of FMD among COVID-19 patients, as well as to further determine the mechanisms involved.

Telomerase Reverse Transcriptase Mediates Restoration of Functional Vasodilation in Isolated Coronary Microvessels of Aged Female Rats

IntroductionReactive oxygen species (ROS) increase during aging, resulting in decreased NO signaling and the production of ROS damages cellular components including mitochondrial DNA. Telomerase Reverse Transcriptase (TERT) has ROS‐suppressing effects in the mitochondria and is decreased in coronary artery disease (CAD). Upregulation of Telomerase restores NO mediated vasodilation in CAD. We hypothesize that treatment of isolated coronary microvessels of aged female rats with peptide nucXTERT (shunts TERT to mitochondria) will increase functional vasodilation while treatment of isolated coronary microvessels from young female rats with mitoXTERT (shunts TERT away from mitochondria) will blunt functional vasodilation.MethodsIsolated coronary microvessels from young (4 months, n = 2) or old (24 months, n = 5) Fischer 344 female rats were assessed via pressure myography. Vessels were incubated for 12 hours with nucXTERT or mitoXTERT, AGS 499 (TERT activator), or a scramble peptide negative control. Vessel relaxation to dobutamine (10−5 – 10−10 M), isoproterenol (10−4 – 10−8 M), acetylcholine (10−4 – 10−9 M), and H2O2 (10−3 – 10−8 M) was assessed post‐incubation.ResultsnucXTERT and AGS 499 treated vessel percent relaxation in old rats for isoproterenol, acetylcholine, and dobutamine were increased compared to scramble treated vessels (Figure 1a–c). Compared to scramble, nucXTERT increase of percent relaxation was significant for isoproterenol at 10−4 – 10−6 M (p = .007, .003, and .013, respectively), dobutamine at 10−5 – 10−7 M (p = &lt; .001, .016, and .016, respectively) and near significant for acetylcholine at 10−4 and 10−6 M (p = .097 and .062, respectively). Following nucxTERT and AGS 499 treatment, the percent relaxation to H2O2 (7.3% and 3.8% at max dilation, respectively) was reduced compared to scramble‐treated vessels (34.5% at max dilation)(Figure 1d). As expected, mitoXTERT incubation of young rat microvessels abrogated vasodilation of vessels to isoproterenol, acetylcholine, and dobutamine compared to scramble and AGS 499 responses. Hydrogen peroxide percent relaxation was increased in mitoXTERT (83.9% at max dose) compared to AGS 499 and scramble (3.79% and 34.38%, respectively).ConclusionsThe data support a vasoprotective and restorative role of TERT in coronary microvessels from aged female rats as adrenergic and acetylcholine‐mediated vasodilation were increased in nucXTERT and AGS 499 relative to the scrambled peptide. With nucXTERT, TERT was being blocked from nuclear translocation, increasing extranuclear TERT available for mitochondrial protection from ROS. Shunting TERT away from the mitochondria via mitoXTERT blunted adrenergic and acetylcholine‐mediated vasodilation as less TERT was available for mitochondrial ROS protection. Considering oxidative stress is a driver of CAD and the concomitant importance of altering vessel patency to allow for adequate blood flow to match metabolic demand, the modulation of TERT may be a holistic and plausible future clinical direction.Support or Funding InformationFunding for this project was provided by NIH RO1 AG053585 and the Gheen’s Foundation to Amanda Jo LeBlanc.Old rat isolated coronary arteriole vasoreactivity with nucXTERT peptide, AGS 499, or scramble peptide incubation. Shown as percent relaxation to isoproterenol (a) dobutamine (b) acetylcholine (c) and hydrogen peroxide (d). p ≤ 0.05 for nucXTERT (*) vs scramble. Data are presented as means ± SE, analyzed with two‐way repeated measures ANOVA followed by post‐hoc Bonferroni test.Figure 1

Elucidation of Vasodilation Response and Structure Activity Relationships of N²,N⁴-Disubstituted Quinazoline 2,4-Diamines in a Rat Pulmonary Artery Model.

Pulmonary arterial hypertension (PAH) is a rare and progressive disease arising from various etiologies and pathogenesis. PAH decreases life expectancy due to pulmonary vascular remodeling, elevation of mean pulmonary arterial pressure, and ultimately progresses to heart failure. While clinical treatments are available to reduce the associated symptoms, a complete cure has yet to be found. Phosphodiesterase-5 (PDE-5) inhibition has been identified as a possible intervention point in PAH treatment. The functional vasodilation response to N2,N4-diamino quinazoline analogues with differing PDE-5 inhibitory activities and varying physicochemical properties were assessed in both endothelium-intact and denuded rat pulmonary arteries to gain greater insight into their mode of action. All analogues produced vasorelaxant effects with EC50s ranging from 0.58 ± 0.22 µM to ˃30 µM. It was observed that vasodilation response in intact vessels was highly correlated with that of denuded vessels. The ~10% drop in activity is consistent with a loss of the nitric oxide mediated cyclic guanosine monophosphate (NO/cGMP) pathway in the latter case. A moderate correlation between the vasodilation response and PDE-5 inhibitory activity in the intact vessels was observed. Experimental protocol using the alpha-adrenergic (α1) receptor agonist, phenylephrine (PE), was undertaken to assess whether quinazoline derivatives showed competitive behavior similar to the α1 receptor blocker, prazosin, itself a quinazoline derivative, or to the PDE-5 inhibitor, sildenafil. Competitive experiments with the α1-adrenergic receptor agonist point to quinazoline derivatives under investigation here act via PDE-5 inhibition and not the former. The pre-incubation of pulmonary arterial rings with quinazoline test compounds (10 μM) reduced the contractile response to PE around 40–60%. The most promising compound (9) possessed ~32 folds higher selectivity in terms of vasodilation to its mammalian A549 cell cytotoxicity. This study provides experi0 0mental basis for PDE-5 inhibition as the mode of action for vasodilation by N2,N4-diamino quinazoline analogues along with their safety studies that may be beneficial in the treatment of various cardiovascular pathologies.

Effects of initial treatment on the dynamics of renal functional reserve in stage-I hypertensive patients

Objective: To determine the functional state of the kidneys in patients with the I stage essential hypertension with different total risk of cardiovascular complications and to assess the effect of early medication (angiotensin II receptor blocker) on the dynamics of changes in the functional renal reserve. Material and Methods: This study included 80 patients with the established diagnosis - the I stage essential hypertension of the I-III grade in age from 18 to 60 years (28 women (35 %), the average age is 40,1 (35,0-56,0) years; men - 57 (71%), average age – 40,2 (37,0-53,0) years. A risk factor count was used to assess the overall risk of cardiovascular complications in hypertension. Functional renal reserve and endothelium-independent vasodilation of the brachial artery were defined. Statistical analysis was performed using SPSS Statistics. Results: In patients with high risk, the depleted and decreased functional renal reserve was most often detected, which was 55% of patients. It is also important to note that patients with even a low risk of cardiovascular complications have already had renal damage as a reduced or depleted functional renal reserve. Patients with a low risk of cardiovascular complications who had not started drug treatment on their first visit had a 2.3-fold (95% CI 1,048-4,896) higher relative risk of developing kidney damage in comparison with patients who had started medication therapy without delay. Conclusion: Diagnosis of functional renal reserve in patients with a low risk of developing cardiovascular complications in stage I arterial hypertension is an important component for predicting the further development of kidney damage and timely prevention of its progression. The early onset of drug treatment reduces the relative risk of developing kidney damage by 2.3 times in patients with arterial hypertension.

Recovery of Functional Vasodilation During Skeletal Muscle Regeneration

Functional vasodilation underlies the increase in skeletal muscle blood flow to supply the metabolic demands of contracting myofibers. Following injury, myofibers degenerate and then regenerate through satellite cell activation, thereby recapitulating myogenesis. While the microvasculature is also disrupted, the recovery of functional vasodilation during myofiber regeneration is unexplored. The goal of this study was to evaluate the time course and magnitude of the recovery of functional vasodilation in microvascular resistance networks at defined time points during skeletal muscle regeneration (confirmed with histology of muscle cross‐sections). In male C57BL/6J mice (4 months old; n = 5 per group), the gluteus maximus muscle (GM) was injured by local injection of barium chloride solution (1.2% solution, 75 μl). At 5, 10, 21 and 35 days (d) post injury, the resistance network of the GM was evaluated (versus Control) in feed arteries (FA), first‐ (1A), second‐ (2A) and third‐order (3A) arterioles using intravital microscopy in anesthetized mice (pentobarbital sodium; 60 mg/kg). For each branch order, maximal internal diameter (ID; topical sodium nitroprusside, 10−4 M) was not different through 35d (mean values: FA, ~62 μm; 1A, ~55 μm; 2A, ~43 μm; 3A, ~30 μm). Under resting conditions, the ID of all branch orders was greater than Control at 5d and remained elevated in FA at 10d (P&lt;0.05). Respective resting IDs recovered to Control at 21d (FA, ~45 μm; 1A, ~32 μm; 2A, 22 μm; 3A, ~11 μm). Thus, while spontaneous vasomotor tone was initially attenuated throughout the network, it recovered by 21d in all branch orders (FA, ~27%; 1A, ~40%; 2A, ~50%; 3A, 63%). Steady state vasodilation to rhythmic twitch contractions (4 Hz, 30s) was impaired at 5d (with degenerated myofibers), attenuated at 10d (primarily new myofibers), improved through 21d (maturing myofibers) and by 35d was not different from Control. Rapid onset vasodilation to tetanic contraction (100 Hz, 500 ms) was also impaired at 5d, attenuated at 10d (P&lt;0.05), improved through 21d and by 35d was not different from Control across respective branch orders. We suggest that elevated resting diameters with diminished vasomotor tone during the initial stages of regeneration promote tissue perfusion as myofiber recovery begins. During maturation of nascent myofibers, restoration of vasomotor tone underscores the recovery of functional vasodilation to both rhythmic and tetanic contractions. Nevertheless, vasomotor tone recovers sooner than the ability of regenerating myofibers to elicit functional vasodilation.Support or Funding InformationAHA 17PRE33410260, NIH R37 HL041026This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Impact of Primary Myoblast Transplantation on Functional Vasodilation Following Arteriogenesis in Mice with Diet‐Induced Obesity

Peripheral arterial occlusive disease (PAOD) is characterized by reduced blood flow to the lower limbs, causing claudication and tissue loss. Proximal collateral arteries are natural bypasses to atherosclerotic occlusions in major arteries. A variety of cell‐based therapies have been tested clinically to enhance collateral arteriogenesis. Unfortunately, their efficacy has been poor, despite promising results from early clinical and preclinical studies, possibly because most preclinical studies are performed with mice that lack co‐morbidities present in most patients that are known to impair arteriogenesis. Therefore, additional cell‐types must be explored in animal models that more closely recapitulate the clinical phenotype to determine if pro‐regenerative therapies are a viable treatment for PAOD. Myogenic cells are an attractive candidate, given their co‐localization with the skeletal muscle vasculature niche and their ability to enhance angiogenesis. Therefore, we tested the hypothesis that primary myoblasts would rescue functional vasodilation in the superficial gracilis collateral following arteriogenesis in mice with diet‐induced obesity (DIO).In these studies, C57Bl/6 mice were fed a high‐fat diet from 6‐weeks of age; experiments were performed at about 4‐months of age. First, we performed a pilot study to evaluate functional vasodilation (vasodilation in response to electrically‐stimulated gracilis muscle contraction) in unoperated animals. As expected, vasodilation was impaired in the right (42±4 vs. 58±4 μm) and left (40±5 vs. 52±5 μm), as compared to lean controls.Having established impaired vasodilation in mice with DIO, we ligated the femoral artery in mice to model obesity and the chronic ischemia aspect of PAOD and measured functional vasodilation at day‐7 following surgery. Pilot data suggests that functional vasodilation is reduced in mice with DIO at day‐7 following surgery (resting: 80±10 μm, dilated: 82±8 μm) and that cell transplantation appears to enhance arteriogenesis and improve functional vasodilation (resting: 93±8 μm, dilated: 108±8 μm). Additional experiments are necessary to confirm these findings, but the data suggest that myoblasts strengthen the arteriogenic response. Future studies may involve additional time points to investigate the time course and duration of myoblast‐rescued arteriogenesis and functional vasodilation following arterial occlusion.Support or Funding InformationResearch was funded by the California Institute of Regenerative Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanoadaptation, arteriogenesis, and vascular reactivity in male and female C57Bl/6 mice

Women have a higher incidence of peripheral arterial occlusive disease (PAOD) and a greater likelihood for developing critical limb ischemia. The importance of the collateral circulation in maintaining tissue blood flow following arterial occlusion suggests that this sexually‐dimorphic response may be due to sex differences in collateral development and function. In experimental animals, females exhibit impaired regional recovery (i.e. limb pressure or resistance) that may not may not require estrogen deficiency. Although the collateral circulation is the primary site of blood flow resistance following arterial occlusion and the likely explanation of any impairment in hindlimb recovery, neither of these conflicting reports directly evaluated collateral enlargement or function. Therefore, we tested the hypothesis that collateral enlargement and reactivity were impaired in female mice.All experiments were performed in C57Bl/6 mice at day‐7 or day‐28 following femoral artery ligation (FAL), between the epigastric/profunda and popliteal branches. In the stem to the collateral circulation (i.e. the profunda femoris), enlargement does not involve cellular proliferation, so we evaluated mecahnoadaptive outward remodeling by measuring vascular smooth muscle cell length and overlap with immunofluorescence and confocal microscopy. At day‐7 following FAL, vascular smooth muscle cell (VSMC) length increased 25% in both male and female mice. Similarly, VSMC overlap decreased by 30% in both male and female mice‐indicating roughly equivalent mechanoadaptive outward remodeling. At the midzone of the gracilis anterior collateral, inner and outer diameter increased 100% in both males and females, while wall thinkness increased over 200%, indicating similar levels of hypertrophic outward remodeling in both sexes. Given the lack of difference in enlargement between male and female collaterals, we next evaluated reactivity, to determine if female mice exhibit impaired collateral function. As expected, reactivity was substantially impaired at day‐7 following FAL, with collateral midzone diameter increasing 15% during functional vasodilation, as compared to nearly 60% increases in sham‐operated contralateral collaterals; responses were not different between males and females. At day‐28 following FAL, collateral midzone reactivity in males was restored, and no longer different from the contralateral control, with both increasing 75% during functional vasodilation. Surprisingly, collateral midzone reactivity was enhanced in females, as compared to both males and the contralateral control, increasing 150% during functional vasodilation.If these data are representative of all collateral vessels, they suggest that impairments in flow recovery following arterial occlusion in female mice with intact ovaries are likely due to dysfunction in the distal microvasculature. Since female mice exhibit more impaired vasodilation during hypercholesterolemia, we will evaluate the collateral enlargement and function in a more clinically‐relevant animal model with vascular disease risk factors in future animal studies aimed at understanding the worse prognosis of females patients with PAOD.

Sympathetic escape in a theoretical model of microvascular flow regulation (1079.18)

A mathematical simulation of flow regulation is used to investigate the hypothesis that vasoconstriction due to sympathetic nerve activity (SNA) in smaller arterioles is inhibited by functional vasodilation at higher values of oxygen demand, termed functional sympatholysis. A network with 13 vessel compartments in series is used, each compartment representing different size ranges of arterioles and venules. The model includes 5 actively regulating arteriolar segments with diameters influenced by shear stress, wall tension, metabolic regulation, and SNA. Metabolic signals are assumed to be propagated upstream along vessel walls via a conducted response. We show that for appropriately chosen parameter values, the vasoconstriction due to SNA in feed arterioles overcomes metabolic vasodilation at higher oxygen demand, resulting in persistent vasoconstriction throughout the period of SNA. In smaller arterioles under similar conditions, however, the initial vasoconstriction with SNA is followed by partial relaxation, consistent with the experimentally observed phenomenon of sympathetic escape. This implies that metabolic responses can account for observed characteristics of functional sympatholysis, including the different responses of large and small arterioles.Grant Funding Source: Supported by NIH grant HL070657.

Impaired Vascular KATP Function Attenuates Exercise Capacity in Obese Zucker Rats

Obese subjects exhibit decreased exercise capacity (VO2max ). We have shown that vascular KATP channel mediates arteriolar dilation to muscle contraction. We hypothesize that exercise capacity is decreased in obesity due to impaired vascular KATP function. The VO2max was measured in LZR and OZR by treadmill running before and following treatment with the KATP blocker glibenclamide i.p. One week later, the spinotrapezius muscle was prepared for in vivo microscopy. Arcade arteriolar diameters were measured following muscle contraction or application of the KATP opener cromakalim before and after glibenclamide application. In additional animals, LZR and OZR were treated with apocynin for five weeks. VO2max and arteriolar dilation experiments were repeated. The OZR exhibited decreased VO2max , functional and cromakalim-induced vasodilation as compared with LZR. Glibenclamide had no effect on VO2max and functional vasodilation in OZR, but significantly inhibited responses in LZR. Vascular superoxide levels and NADPH oxidase activity were increased in OZR, but reduced in apocynin-treated OZR. Apocynin increased the VO2max , functional and cromakalim-induced vasodilation in OZR with no effect in LZR. Exercise capacity is dependent on vascular KATP channel function. The reduced exercise capacity in OZR appears to be due in part to superoxide-mediated impairment in vascular KATP function.

Pharmacological Studies of Tentacle Extract from the Jellyfish Cyanea capillata in Isolated Rat Aorta

Our previous studies demonstrated that tentacle extract (TE) from the jellyfish, Cyanea capillata, could cause a dose-dependent increase of systolic blood pressure, which seemed to be the result of direct constriction of vascular smooth muscle (VSM). The aim of this study is to investigate whether TE could induce vasoconstriction in vitro and to explore its potential mechanism. Using isolated aorta rings, a direct contractile response of TE was verified, which showed that TE could induce concentration-dependent contractile responses in both endothelium-intact and -denuded aortas. Interestingly, the amplitude of contraction in the endothelium-denuded aorta was much stronger than that in the endothelium-intact one, implying that TE might also bring a weak functional relaxation in addition to vasoconstriction. Further drug intervention experiments indicated that the functional vasodilation might be mediated by nitric oxide, and that TE-induced vasoconstriction could be attributed to calcium influx via voltage-operated calcium channels (VOCCs) from the extracellular space, as well as sarcoplasmic reticulum (SR) Ca2+ release via the inositol 1,4,5-trisphosphate receptor (IP3R), leading to an increase in [Ca2+]c, instead of activation of the PLC/DAG/PKC pathway or the sympathetic nerve system.

Functional Vasodilation is Impaired in Arterialized Capillaries following Ischemia

Ischemic disease is the leading cause of morbidity and mortality in the developed world. Patients with a well‐developed collateral network experience less severe symptoms following ischemic events, but not all patients have well‐developed collateral networks. Stimulating arterialization of capillaries into functional collaterals could minimize the impact of ischemic events in patients with poorly‐developed collateral networks. Stable arterialized capillaries develop following occlusion, but the reactivity of these vessels is unknown. Ischemic events, such as angina, occur with increased metabolic demand, so effective collaterals must be capable of dilating and increasing blood flow to support tissue oxygen demand. To examine the ability of arterialized capillaries to regulate blood flow, we ligated the lateral cranial feed artery of the right spinotrapezius muscle and measured functional vasodilation at days 7 and 21 following arterial occlusion. Vasodilation in arterialized capillaries was absent at day 7 compared to arterioles with a similar resting diameter. (2% ± 2% vs. 29% ± 12% p&lt; 0.05) Vasodilation was restored by day 21.(167% ± 17% vs 81% ± 13% p&gt;; 0.05) Studies are currently underway to examine the mechanism of impaired vasodilation at day 7 and the hemodynamic effects of vasodilation at day 21 following arterial occlusion.

The impact of outward remodeling on vasodilation in skeletal muscle resistance arteries

Collateral enlargement following arterial occlusion is thought to rescue downstream tissues from severe ischemic insult in patients with ischemic disease, such as peripheral arterial occlusive disease. However, in animal models, collateral‐dependent hyperemia is reduced after arterial occlusion, suggesting that vasodilation may be impaired. Impaired vasodilation would compromise the effectiveness of collaterals, as ischemia occurs during periods of increased metabolic demand. To assess the impact of collateral enlargement on vasodilation in resistance arteries, we ligated the femoral arteries of young, otherwise healthy mice to induce outward remodeling in the collateral circuit. Outward remodeling transiently impairs functional vasodilation, which is reduced at day‐7 (62 ± 3% vs. 93 ± 10%, p &lt; 0.05), but improves by day‐28 (101 ± 4% vs. 115 ± 10%). Vascular reactivity studies indicate the impaired vasodilation is due to impaired smooth muscle‐dependent responses during outward remodeling, as both endothelial‐ and smooth muscle‐dependent vasodilation is impaired at day‐7 (100 ± 16% vs. 149 ± 15%, p &lt; 0.05 and 103 ± 15% vs. 125 ± 15%, p &lt; 0.05, respectively), but not at day‐28 (136 ± 24% vs. 142 ± 20% and 146 ± 18% vs. 124 ± 10%). Studies examining the cellular mechanism of the day‐7 impairment and the impact of impaired vasodilation on hemodynamics are currently underway.

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Improved functional vasodilation in obese Zucker rats following exercise training

Obese individuals exhibit impaired functional vasodilation and exercise performance. We have demonstrated in obese Zucker rats (OZ), a model of morbid obesity, that insulin resistance impairs functional vasodilation via an increased thromboxane receptor (TP)-mediated vasoconstriction. Chronic treadmill exercise training improves functional vasodilation in the spinotrapezius muscle of the OZ, but the mechanisms responsible for the improvement in functional vasodilation are not clear. Based on evidence that exercise training improves insulin resistance, we hypothesized that, in the OZ, exercise training increases functional vasodilation and exercise capability due to decreases TP-mediated vasoconstriction associated with improved insulin sensitivity. Six-week-old lean Zucker rats (LZ) and OZ were exercised on a treadmill (24 m/min, 30 min/day, 5 days/wk) for 6 wk. An oral glucose tolerance test was performed at the end of the training period. We measured functional vasodilation in both exercise trained (spinotrapezius) and nonexercise trained (cremaster) muscles to determine whether the improved functional vasodilation following exercise training in OZ is due to a systemic improved insulin resistance. Compared with LZ, the sedentary OZ exhibited impairments in glucose tolerance and functional vasodilation in both muscles. The TP antagonist SQ-29548 improved the vasodilator responses in the sedentary OZ with no effect in the LZ. Exercising training of the LZ increased the functional vasodilation in spinotrapezius muscle, with no effect in the cremaster muscle. Exercising training of the OZ improved glucose tolerance, along with increased functional vasodilation, in both the spinotrapezius and cremaster muscles. SQ-29548 treatment had no effect on the vasodilator responses in either cremaster or spinotrapezius muscles of the exercise-trained OZ. These results suggest that, in the OZ, there is a global effect of exercising training to improve insulin resistance and increase functional vasodilation via a decreased TP-mediated vasoconstriction.

Arteriogenesis and ischemia impair functional vasodilation in resistance arteries

Collateral-dependent hyperemia is reduced following arterial occlusion, but the role of impaired vasodilation in this reduction is unknown. Additionally, ischemia impairs arteriolar vasodilation, but its impact on resistance artery vasodilation is unknown. To examine the impact of arteriogenesis and ischemia on resistance artery vasodilation, we ligated or resected the femoral artery, respectively. Arteriogenesis transiently impairs functional vasodilation, which is reduced at day-7, but improves by day-14. We hypothesize that the presence of synthetic smooth muscle cells underlies this impairment; studies are underway to examine smooth muscle cell phenotype during arteriogenesis. Ischemia also impairs functional vasodilation by making resistance arteries less sensitive to vasodilation stimuli- vasodilation occurs in response to 500μs pulse durations, but not 200μs at day-14. This impairment seems to be exacerbated with age. Although the dynamic range of acetylcholine-mediated vasodilation is reduced in ischemic arteries, the maximum diameter is not different from control. Studies are underway to determine if ischemic arteries have undergone arteriogenesis or are dilated at rest due to tissue hypoxia.

Chronic Hindlimb Ischemia Impairs Functional Vasodilation and Vascular Reactivity in Mouse Feed Arteries

Vasodilation of lower leg arterioles is impaired in animal models of chronic peripheral ischemia. In addition to arterioles, feed arteries are a critical component of the vascular resistance network, accounting for as much as 50% of the pressure drop across the arterial circulation. Despite the critical importance of feed arteries in blood flow control, the impact of ischemia on feed artery vascular reactivity is unknown. At 14 days following unilateral resection of the femoral–saphenous artery–vein pair, functional vasodilation of the profunda femoris artery was severely impaired, 11 ± 9 versus 152 ± 22%. Although endothelial and smooth muscle-dependent vasodilation were both impaired in ischemic arteries compared to control arteries (Ach: 40 ± 14 versus 81 ± 11%, SNP: 43 ± 12 versus and 85 ± 11%), the responses to acetylcholine and sodium nitroprusside were similar, implicating impaired smooth muscle-dependent vasodilation. Conversely, vasoconstriction responses to norepinephrine were not different between ischemic and control arteries, −68 ± 3 versus −66 ± 3%, indicating that smooth muscle cells were functional following the ischemic insult. Finally, maximal dilation responses to acetylcholine, ex vivo, were significantly impaired in the ischemic artery compared to control, 71 ± 9 versus 97 ± 2%, despite a similar generation of myogenic tone to the same intravascular pressure (80 mmHg). These data indicate that ischemia impairs feed artery vasodilation by impairing the responsiveness of the vascular wall to vasodilating stimuli. Future studies to examine the mechanistic basis for the impact of ischemia on vascular reactivity or treatment strategies to improve vascular reactivity following ischemia could provide the foundation for an alternative therapeutic paradigm for peripheral arterial occlusive disease.

Impaired vascular reactivity following chronic ischemia in arteries of the mouse hindlimb

Collateral‐dependent hyperemia is reduced following chronic ischemia, but the contribution of individual vessel reactivity is unknown. Further, it is not known what aspect of the complex ischemic injury response impacts vascular reactivity. To determine the impact of ischemia on vascular reactivity, we measured functional vasodilation in the muscular branch artery following resection of the femoral artery proximal to the muscular branch, which induces ischemia in the muscular branch artery. On day‐14 after surgery the diameter of the muscular branch was measured using side‐stream dark field (SDF) imaging intravital microscopy. At moderate intensity skeletal muscle contraction (1mA, 200μs, 8Hz, 90sec), functional vasodilation is reduced compared to the contralateral limb (10.84 ± 8.79% versus 151.70 ± 22.15% increase above baseline). Conversely, at high intensity skeletal muscle contraction (1mA, 500μs, 8Hz, 90sec), the initial vasodilation is not different from the contralateral limb, but the ischemic arteries are refractory to regaining their resting diameter. The control artery returned to baseline diameter within 10‐minutes following high‐intensity muscle contraction, while the ischemic artery did not return to baseline after 25 minutes. Further investigation is underway to determine if endothelial or smooth muscle dysfunction underlies this abnormal reactivity in ischemic arteries.

Adipocyte-derived factor reduces vasodilatory capability in ob−/ob− mice

Obesity is associated with impaired functional hyperemic response. We have shown that ATP-sensitive potassium (K(ATP)) channels are important in mediating functional vasodilation. Adipocyte-derived factors (ADFs) can alter vascular tone via opening K(ATP) channels. We hypothesize that, in an animal model of obesity, ADFs will decrease basal arteriolar tone by opening K(ATP) channels, resulting in an attenuated functional vasodilation. We used wild-type (WT) mice and ob(-)/ob(-) mice (ob) to test this hypothesis. The spinotrapezius muscle was prepared for the microcirculatory observation of arcade arterioles, and we measured the vasodilatory responses to muscle stimulation. The basal arteriolar diameter was larger in ob mice compared with WT mice. The K(ATP) channel inhibitor glibenclamide (10 microM) decreased arteriolar diameter in ob mice with no effect in WT mice. The increase in arteriolar diameter induced by muscle stimulation was attenuated in ob mice compared with WT mice. To determine the mechanisms for the opening of K(ATP) channels, fat was collected from the ob mice, subcutaneous fat from around the spinotrapezius muscle (OBSF) or visceral fat (OBVF) and was incubated in physiological saline solution (PSS). The vasodilatory responses to the fat-conditioned PSS were determined in WT mice. Treatment with OBSF- or OBVF -conditioned PSS increased the arteriolar diameters in WT mice, a dilation that was inhibited by glibenclamide. The absolute diameters induced by muscle stimulation were not altered by the fat-conditioned PSS. These results suggest that, in ob mice, local ADFs reduce the functional vasodilatory capability via opening K(ATP) channels.