Perfusion Ratio Research Articles

Endothelial smoothened-dependent hedgehog signaling is not required for sonic hedgehog induced angiogenesis or ischemic tissue repair

Sonic Hedgehog (Shh) signaling induces neovascularization and angiogenesis. It is not known whether the hedgehog signaling pathway in endothelial cells is essential to angiogenesis. Smoothened (Smo) transduces hedgehog signaling across the cell membrane. This study assessed whether endothelial Smoothened-dependent Shh signaling is required for Shh-mediated angiogenesis and ischemic tissue repair. Endothelial-specific smoothened knockout mice, eSmoNull were created using Cre-lox recombination system. eSmoNull mice had no observable phenotype at baseline and showed normal cardiac function. Smoothened in CD31+ cells isolated from eSmoNull hearts was significantly reduced compared to CD31+ cells from eSmoWT littermate control hearts. Fluorescence immunostaining of eSmoNull heart sections showed Smo expression in endothelial cells was abolished. The hind-limb ischemia (HLI) model was used to assess the response to ischemic injury. Perfusion ratio, limb motor function, and limb necrosis were not significantly different after HLI between eSmoNull mice and eSmoWT. Capillary densities in the ischemic limb in eSmoNull mice were also similar to eSmoWT at 4 weeks after HLI. Next, response to exogenous Shh was assessed in the corneal angiogenesis model. There was no significant difference in corneal angiogenesis induced by administration of Shh pellets between eSmoWT and eSmoNull mice. Furthermore, in vitro experiments demonstrated that direct Shh had limited effects on endothelial cell proliferation and migration. However, conditioned media from Shh-treated fibroblasts had a more potent effect on endothelial cell proliferation and migration than non-treated conditioned media. Furthermore, Shh treatment of fibroblasts dramatically stimulated angiogenic growth factor expression, including PDGF-B, VEGF-A, HGF and IGF. PDGF-B was the most upregulated and may contribute to the large neo-vessels associated with Shh-induced angiogenesis. Taken together, these data demonstrate that Shh signaling via Smoothened in endothelial cells is not required for angiogenesis and ischemic tissue repair. Shh signaling via stromal cells likely mediates its angiogenic effects.

Myocardial perfusion quantification using simultaneously acquired 13 NH3 -ammonia PET and dynamic contrast-enhanced MRI in patients at rest and stress.

Systematic differences with respect to myocardial perfusion quantification exist between DCE-MRI and PET. Using the potential of integrated PET/MRI, this study was conceived to compare perfusion quantification on the basis of simultaneously acquired 13 NH3 -ammonia PET and DCE-MRI data in patients at rest and stress. Twenty-nine patients were examined on a 3T PET/MRI scanner. DCE-MRI was implemented in dual-sequence design and additional T1 mapping for signal normalization. Four different deconvolution methods including a modified version of the Fermi technique were compared against 13 NH3 -ammonia results. Cohort-average flow comparison yielded higher resting flows for DCE-MRI than for PET and, therefore, significantly lower DCE-MRI perfusion ratios under the common assumption of equal arterial and tissue hematocrit. Absolute flow values were strongly correlated in both slice-average (R2 = 0.82) and regional (R2 = 0.7) evaluations. Different DCE-MRI deconvolution methods yielded similar flow result with exception of an unconstrained Fermi method exhibiting outliers at high flows when compared with PET. Thresholds for Ischemia classification may not be directly tradable between PET and MRI flow values. Differences in perfusion ratios between PET and DCE-MRI may be lifted by using stress/rest-specific hematocrit conversion. Proper physiological constraints are advised in model-constrained deconvolution.

Myocardial perfusion at rest in uncomplicated type 2 diabetes patients without coronary artery disease evaluated by 320-multidetector computed tomography: A pilot study.

Using computed tomography myocardial perfusion imaging (CTP) to investigate resting myocardial perfusion alterations in uncomplicated type 2 diabetes mellitus (T2DM) patients without obstructive coronary artery disease (CAD).A total of 34 participants with 544 myocardial segments were included prospectively: 17 uncomplicated T2DM patients with no significant coronary artery stenosis on coronary computed tomography angiography and 17 healthy controls. Myocardial perfusion was evaluated by transmural perfusion ratio (TPR). Parameters of cardiac structure and function were measured for cardiac comprehensive assessment. Analyses included descriptive statistics and group comparisons.TPR of segments 5, 7, 9, 10 to 14 were significantly reduced in T2DM group compared with controls (P < .05). When 16 myocardial segments were localized into different areas according to the wall orientations, axial levels of left ventricle and coronary artery territories, respectively, TPR of each area in T2DM group were significantly lower than those in the control group (P < .05). No significant differences were found in cardiac anatomy and function analyses between 2 groups.In uncomplicated T2DM patients without obstructive CAD, myocardial perfusion impairments were present and may develop prior to cardiac morphological and functional abnormalities, which can be early detected by CTP.

Arterial Spin Labeling Cerebral Perfusion Magnetic Resonance Imaging in Migraine Aura: An Observational Study

Changes in cerebral perfusion during migraine with aura (MA) have been assessed mainly using dynamic susceptibility contrast (DSC) magnetic resonance perfusion imaging. A contrast agent-free method to assess these changes would be desirable. We assessed changes in cerebral perfusion during MA using arterial spin labeling (ASL) perfusion magnetic resonance imaging. We investigated 4 patients with a standardized protocol including ASL perfusion imaging during MA (n = 2) or early headache phase (n = 2) and asymptomatic follow-up. Semiquantitative evaluation was done using a region of interest (ROI) within hypoperfused or hyperperfused areas and corresponding ROIs in the contralateral hemisphere. Relative ratios of mean perfusion in the corresponding ROIs were calculated. DSC imaging was done at initial time points and compared visually with ASL findings. In all patients, regional perfusion changes were detected in the acute phase. These abnormalities did not respect the boundaries of major cerebral vascular territories but overlapped onto adjoining regions. During MA, adjacent hypoperfused and hyperperfused areas were found, whereas during headache, regional hyperperfusion only was observed. Perfusion abnormalities normalized on follow-up. ASL perfusion imaging is a contrast agent-free method suitable for assessment of reversible perfusion changes during or immediately after MA.

Pulsatility Index as a Novel Parameter for Perfusion in Mouse Model of Hindlimb Ischemia

Background/Aims: In clinical settings, the pulsatility index (PI) has become a widely used tool for monitoring obstetrics or other vascular diseases. It is based on the maximum Doppler shift waveform derived from ultrasonography. However, it remains unclear whether the PI levels are correctly predicted from the perfusion in mouse model of hindlimb ischemia. Methods: To explore the relationship between PI and perfusion, we generated a unilateral hindlimb ischemia model in 8-week-old C57BL/6 male mice by ligation of the right common iliac artery and femoral artery. These mice were monitored with laser Doppler perfusion imaging (LDPI) and an ultrasound system (Vevo2100). Vessel densities in ischemic skeletal muscles were measured with vWF staining, which functions as a marker for endothelial cells. In order to further verify PI evaluation in other conditions, we performed therapeutic experiments using hindlimb ischemic mouse with PBS or FGF2 treatment. Results: In the mouse model of hindlimb ischemia, the PI levels were continuously elevated and were accompanied by an increased ratio of perfusion to blood flow. 1 and 4 weeks after ischemia, the densities of vWF staining were correlated with PI values. Moreover, the PI index exactly reflected the perfusion in hindlimb ischemic mice after FGF2 treatment, while it indicated the condition of angiogenesis after therapeutic treatment based on the association between PI values and the number of vWF-positive stained cells in muscles. Conclusion: This study confirms the utility of a noninvasive and reproducible ultrasound index for a rapid evaluation of perfusion and blood recovery after hindlimb ischemia in vivo. PI, as one stable and comparable parameter, is correlated with angiogenesis in hindlimb ischemic mouse. Moreover, PI can exactly reflect perfusion and angiogenesis in therapeutic hindlimb ischemic mouse models. This study suggested that PI can serve as a novel index for relatively reproducible and repeatable blood flow recovery in the evaluation of emerging ischemic therapies and disease development in mouse models of hindlimb ischemia.

Evaluation of differential pulmonary perfusion using 99mTc macroaggregated albumin after the Fontan procedure.

The Fontan procedure [total cavopulmonary connection (TCPC)] is the final palliation for patients with univentricular physiology. We studied differential perfusion ratio and percentage uptake of a radiotracer in different zones of each lung following TCPC. Between July 2015 and June 2017, 45 patients underwent 99mTc macroaggregated albumin lung perfusion scan at a mean follow-up period of 49.3 ± SD 26.1 days following TCPC. Differential perfusion ratio and percentage uptake of the radiotracer in the upper, middle and lower zones of each lung were calculated. Post-foot injection [inferior vena cava (IVC) injection], preferential flow to the lungs was as follows: left lung (n = 13, 30.2%), right lung (n = 13, 30.2%) and uniformly to both lungs (n= 17, 39.6%). Post-arm injection [superior vena cava (SVC) injection], preferential flow to the lungs was as follows: left lung (n = 13, 30.2%), right lung (n = 22, 51.2%) and uniformly to both lungs (n= 8, 18.6%). The middle zone was perfused the most in both lungs. Total lower zone mean perfusion was higher than the upper zone following both SVC injection and IVC injection (34.1 ± SD 5.3% vs 17. ± SD 4.1% and 33 ± SD 5.0% vs 17.5 ± SD 4.1%, respectively). In patients with bilateral SVC, post-IVC injection, 6 (75%) patients had preferential flow to the right lung, whereas post-SVC injection, preferential flow to the left lung was visualized in 7 (87.5%) patients. Following TCPC, IVC blood was distributed uniformly in both lungs. SVC blood preferentially perfused the right lung. The middle zone was perfused the most in both lungs.

Hepatocellular carcinoma treated with sorafenib: Arterial tumor perfusion in dynamic contrast-enhanced CT as early imaging biomarkers for survival

ObjectivesTo investigate whether hepatic perfusion CT yields early imaging biomarkers predictive of the prognosis of hepatocellular carcinoma (HCC) patients treated with sorafenib. MethodsWe evaluated 36 HCC patients who underwent hepatic perfusion CT before- and one week after sorafenib therapy. We measured arterial and portal perfusion in the hepatic tumor and liver parenchyma [(AP)(PP)tumor], [(AP)(PP)liver]. The perfusion ratio was calculated by dividing the post- by the pre-sorafenib value. The effect of each value on the overall survival rate was analyzed with the Cox proportional hazards model; statistically significant parameters were subjected to receiver operating characteristic analysis based on median survival after sorafenib administration to determine the overall survival rate with the Kaplan-Meier method. ResultsPre-APtumor was significantly associated with the overall survival rate (hazard ratio (HR) and 95% confidence interval (CI), 0.16 and 0.02-0.84, p=0.03). The APtumor ratio tended to be associated with the overall survival rate (HR and 95% CI, 2.94 and 0.94-7.88, p=0.06). The overall survival rate was higher in patients with pre-APtumor>71.7mL/min/100mL, and with APtumor ratio≦1.1 (p<0.01 and 0.03, respectively, in Kaplan-Meier method with log-rank). ConclusionHepatic perfusion CT yields early imaging biomarkers for predicting overall survival in HCC patients treated with sorafenib.

Synergistic Therapeutic Effect of Three-Dimensional Stem Cell Clusters and Angiopoietin-1 on Promoting Vascular Regeneration in Ischemic Region.

Peripheral artery disease (PAD) is an ischemic disease characterized by reduced blood flow to the legs, feet, and hands. Human mesenchymal stem cells are an attractive cell source to treat PAD in regenerative medicine. However, in clinical applications, the use of adult stem cells has several limitations, such as low cell viability and low therapeutic efficiency. In this study, we described an innovative method of culturing three-dimensional stem cell clusters (Angiocluster™ [AC]), demonstrated the potential for ACs to differentiate into vascular cells, and assessed the synergistic effects of ACs and angiopoietin-1 (Ang-1) on angiogenesis in ischemic animal models. ACs were formed by culturing human adipose-derived stem cells (hASCs) on a maltose-binding protein-linked basic fibroblast growth factor-immobilized polystyrene surface. ACs released various angiogenic factors, such as vascular endothelial growth factor and interleukin-8, and could differentiate into endothelial lineage cells. However, ACs did not secrete Ang-1, which is an essential component of vascular maturation and anti-inflammation. ACs were combined with Ang-1 and were transplanted into the ischemic lesions of mice for 28 days. Most of the mice receiving the AC + Ang-1 treatment exhibited limb salvage and exhibited similar blood perfusion ratio compared to normal limb. The combination therapy of AC and Ang-1 enhanced angiogenic efficacy by increasing blood vessel regeneration and facilitating the implantation of stem cells into host vessels. Importantly, fibrotic collagen was observed in most of the groups after 28 days of treatment, except for the AC + Ang-1 group. This indicates that the combination therapy is synergistic in minimizing ischemic fibrosis and muscle degeneration. Our results demonstrate that the combination therapy significantly enhanced tissue regeneration and angiogenic efficacy of hASCs and may have wide applications in regenerative medicine.

A pro-angiogenic degradable Mg-poly(lactic-co-glycolic acid) implant combined with rhbFGF in a rat limb ischemia model.

Magnesium (Mg)-based implant has been already used in patients with critical limb ischemia. Site-specific controlled release of recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. We report here on a novel combination implant composed of spiral magnesium and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The preparation method does not involve any complex processes and results in a high encapsulation efficiency (approximately 100%). The degradation of metal Mg raise the microclimate pH in the PLGA polymer, which could well preserve the bioactivity of rhbFGF incorporated in the implant. Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery in rat limb ischemic model. This work marks the first report for controlled release of rhbFGF in combination with metal Mg, and suggests potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia.

Geographic and demographic variabilities of quantitative parameters in stress myocardial computed tomography perfusion.

Background/AimsTo evaluate the geographic and demographic variabilities of the quantitative parameters of computed tomography perfusion (CTP) of the left ventricular (LV) myocardium in patients with normal coronary artery on computed tomography angiography (CTA).MethodsFrom a multicenter CTP registry of stress and static computed tomography, we retrospectively recruited 113 patients (mean age, 60 years; 57 men) without perfusion defect on visual assessment and minimal (< 20% of diameter stenosis) or no coronary artery disease on CTA. Using semiautomatic analysis software, quantitative parameters of the LV myocardium, including the myocardial attenuation in stress and rest phases, transmural perfusion ratio (TPR), and myocardial perfusion reserve index (MPRI), were evaluated in 16 myocardial segments.ResultsIn the lateral wall of the LV myocardium, all quantitative parameters except for MPRI were significantly higher compared with those in the other walls. The MPRI showed consistent values in all myocardial walls (anterior to lateral wall: range, 25% to 27%; p = 0.401). At the basal level of the myocardium, all quantitative parameters were significantly lower than those at the mid- and apical levels. Compared with men, women had significantly higher values of myocardial attenuation and TPR. Age, body mass index, and Framingham risk score were significantly associated with the difference in myocardial attenuation.ConclusionsGeographic and demographic variabilities of quantitative parameters in stress myocardial CTP exist in healthy subjects without significant coronary artery disease. This information may be helpful when assessing myocardial perfusion defects in CTP.

Gamma-variate modeling of indicator dilution curves in electrical impedance tomography.

Electrical impedance tomography (EIT) is a non-invasive imaging technique, that can be used to monitor regional lung ventilation (V̇) in intensive care units (ICU) at bedside. This work introduces a method to extract regional lung perfusion (Q̇) from EIT image streams in order to quantify regional gas exchange in the lungs. EIT data from a single porcine animal trial, recorded during injection of a contrast agent (NaCl 10%) into a central venous catheter (CVC), are used for evaluation. Using semi-negative matrix factorization (Semi-NMF) a set of source signals is extracted from the data. A subsequent non-linear fit of a gamma-variate model to the source signals results in model signals, describing contrast agent flow through the cardio-pulmonary system. A linear fit of the model signals to the EIT image stream then yields functional images ofQ̇. Additionally, a pulmonary transit function (PTF) and parameters, such as mean transit time (MTT), time to peak (TTP) and area under curve (AUC) are derived. In result, EIT was used to track changes of regional lung ventilation to perfusion ratio (V̇/Q̇) during changes of positive end-expiratory pressure (PEEP). Furthermore, correlations of MTT and AUC with cardiac output (CO) indicate that CO measurement by EIT might be possible.

Role of quantitative perfusion scintigraphy evaluation in determination of the efficiency of the procedure before lung volume reduction coil treatment for severe emphysema

Lung volume reduction coil (LVRC) is a new treatment method which exercise capacities among the patients with severe emphysema. In order to determine the emphysema distribution before LVRC treatment, we examined the contribution of combination of the high resolution computed tomography (HRCT) of the lung and the quantitative perfusion scintigraphy to the efficiency of the procedure. The study was conducted retrospectively. The densitometry analysis and scores of emphysema areas of the patients who were found suitable for LVRC treatment in HRCT were determined. Before the procedure, the quantitative lung perfusion scintigraphies were taken. Lobes which had high emphysema score and in which a decrease perfusion was observed in quantitative perfusion scintigraphy were determined as treatment areas. A total of 22 patients were treated. In HRCT it was observed that perfusion decreased in lobes in which the emphysema damage scoring was high. A significant statistical difference was found between pre-and post- procedure measurements in terms of FEV1, RV, and TLC (p<0.05). In six minute walk test, a statistically significant difference was found as 45.4 ± 23.9 m (p= 0.000). No significant difference was determined between perfusion ratios before the procedure and perfusion ratios after the procedure. The quantitative perfusion scintigraphy taken before LVRC treatment in severe emphysema patients guides to determine the target lobe for treatment and makes contribution to the efficiency of the procedure.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia

BackgroundThere is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. MethodsTo determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used—gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. ResultsSignificant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. ConclusionsThe overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

Abstract 320: Delivery of Hepatocyte Growth Factor mRNA From Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease

Biological approaches to augment angiogenesis are promising for treatment of peripheral arterial disease (PAD). We propose the use of scaffold-based modified mRNA (mmRNA) delivery as a favorable approach for transient, localized gene delivery. We hypothesized that hepatocyte growth factor (HGF) mmRNA-seeded nanofibrillar scaffolds will enable localized and temporally controlled delivery of mmRNA, leading to augmentation of angiogenesis in a murine model of PAD. To establish the efficacy of mmRNA therapy, mmRNA encoding green fluorescence protein (GFP) was used as a fluorescent reporter for quantification of transfection efficiency. Aligned nanofibrillar collagen scaffolds were loaded with mmRNA and lipofectamine transfection agent. The temporal kinetics of mmRNA release into media was measured by ribogreen assay. To determine the transfection efficiency, human fibroblasts were cultured on the aligned nanofibrillar scaffolds, or on tissue culture plastic, and the efficiency of transfection was measured for up to 7 days and assayed for GFP expression. Based on ribogreen assay, the cumulative release of GFP mmRNA over the course of 14 days was 235 ng/cm scaffold. In vitro transfection efficiency on aligned scaffolds (75%) was markedly higher than on tissue culture plastic (45%) after 24h. The persistence of cellular transfection as quantified by western blotting showed GFP expression &gt;5 days post-transfection. Next, to demonstrate therapeutic efficacy for treatment of PAD, scaffolds releasing HGF or GFP mmRNA were transplanted to the site of the murine ischemic hindlimb. At the end of the 14 day experiment, laser Doppler spectroscopy showed that HGF mmRNA scaffold group had a higher mean perfusion ratio (0.32 ±0.10) than the GFP mmRNA scaffold group (0.23±0.14), suggesting that HGF-scaffolds improved blood perfusion. In summary, these data suggest that HGF mmRNA-releasing scaffolds marked improved blood perfusion in a murine model of PAD.

Homocysteine-induced peripheral microcirculation dysfunction in zebrafish and its attenuation by L-arginine.

Elevated blood homocysteine (Hcy) level is frequently observed in aged individuals and those with age-related vascular diseases. However, its effect on peripheral microcirculation is still not fully understood. Using in vivo zebrafish model, the degree of Hcy-induced peripheral microcirculation dysfunction is assessed in this study with a proposed dimensionless velocity parameter , where and represent the peripheral microcirculation perfusion and the systemic perfusion levels, respectively. The ratio of the peripheral microcirculation perfusion to the systemic perfusion is largely decreased due to peripheral accumulation of neutrophils, while the systemic perfusion is relatively preserved by increased blood supply from subintestinal vein. Pretreatment with L-arginine attenuates the effects of Hcy on peripheral microcirculation and reduces the peripheral accumulation of neutrophils. Given its convenience, high reproducibility of the observation site, non-invasiveness, and the ease of drug treatment, the present zebrafish model with the proposed parameters will be used as a useful drug screening platform for investigating the pathophysiology of Hcy-induced microvascular diseases.

Endothelial Foxo1 and Foxo3a Synergistically Repress Skeletal Muscle Vascular Growth and Blood Flow Recovery in a Mouse Model of Hind Limb Ischemia.

Neovascularization in peripheral artery disease (PAD) is impaired despite upregulation of factors that promote blood vessel growth which suggest a concomitant increase of anti‐angiogenic repressors. We previously showed that depletion of FoxO proteins (FoxO1/3a/4) induced angiogenesis and enhanced blood flow recovery in a mouse model of PAD. The purpose of this study is to independently assess the roles of endothelial FoxO1 and synergistic actions of FoxO 1 &amp; 3a in a mouse model of PAD. Mice with tamoxifen‐inducible endothelial specific deletions of FoxO1 (PDGFBCreERT2+;FoxO1L/L, FoxO1Δ) and FoxO1/3a (PDGFBCreERT2+;FoxO1/3aL/L, FoxO1/3Δ), or control (PDGFBCreERT2‐, FoxO1L/L) mice, underwent unilateral ligation of the common femoral artery (at ~8–9 weeks of age) to induce a substantial (~80%) reduction of blood flow in the ligated (LIG) compared to the contralateral (CON) leg. Blood flow recovery was analyzed by laser doppler imaging at baseline, then on days 4, 7, 10 and 14 post‐ligation. Muscle histology was conducted after 14 days. FoxO1/3Δ blood flow (LIG/CON leg perfusion ratio) was recovered to basal levels (1.10 ±0.07; n=6) and significantly greater than FoxO1Δ (0.73 ±0.05; P&lt;0.01, n=5) and FoxO1L/L (0.65 ±0.03; P&lt;0.001, n=5) mice 14 days post‐ligation. Neither FoxO1 nor FoxO1/3 deletion altered capillary density (CD) in the control leg. In contrast, CON leg capillary‐to‐fibre ratio (C: F) in FoxO1/3Δ mice (1.02 ±0.04) was significantly lower than FoxO1L/L (1.48 ±0.08) and FoxO1Δ (1.44 ±0.09) mice (P &lt;0.05). Ischemic muscle was characterized by marked cellular infiltration, deposition of fibrous and adipose tissue and myofibre atrophy. Capillary density was significantly increased in ischemic muscles from FoxO1Δ (CON: 746 ±40.15 vs LIG: 1218 ±187.4 capillaries/mm2; P =0.03) and FoxO1/3aΔ (CON: 743.2 ±39.35 vs LIG: 1661 ±158.5 capillaries/mm2; P =0.005) mice. C: F was increased ~25% in ischemic muscle from FoxO1/3Δ mice (1.28 ±0.05; P &lt;0.05) but was unaltered in FoxO1L/L (1.46 ±0.19) or FoxO1Δ mice (1.55 ±0.22). Our data suggest that the combined effects of FoxO1 &amp; 3a repress vascular growth and blood flow recovery in ischemic muscles more than the independent effects of FoxO1. Funded by CIHR.

The Paracrine Effects of Satellite Cells on Collateral Vessel Formation

Satellite cells are myogenic cells that play a critical role in skeletal muscle repair. In healthy muscle they are maintained in a quiescent state but upon injury to the muscle they activate resulting in increased proliferation. While many of the activated satellite cells differentiate into myoblasts which fuse to repair the muscle, some return to the quiescent state to serve as a “stem cell” population. However, another key aspect of muscle regeneration is reestablishing vascular supply, yet the role of satellite cells in this process is not well established. Satellite cells have been shown to produce several proteins that have the potential to act as paracrine signals. Thus, we hypothesized that satellite cells promote vascular growth through paracrine signaling induced by activation following muscle injury or ischemic damage from diseases such as peripheral artery disease. The model of collateral growth used in this study was a murine model of hind limb ischemia in which the femoral artery and vein are ligated and excised. Using this model, we showed ischemic muscle damage increased satellite cell numbers 3.4 fold (p&lt;0.01) as measured by isolating cells from the hind limb muscles using magnetic bead sorting. To determine if satellite cells produce paracrine factors, we used a modified Boyden chamber co‐culture system for migration where satellite cells served as the chemoattractant and proliferation where satellite cells were the growth stimuli. We found that satellite cells freshly isolated from the ischemic limb led to a 3.5 fold increase in smooth muscle cell migration (p&lt;0.0001) and a 1.3 fold increase (p&lt;0.01) in smooth muscle cell proliferation. Additionally, cultured satellite cells increased endothelial cell migration 1.9 fold and proliferation 1.3 fold. Both smooth muscle cell and endothelial cell migration and proliferation are required for the development of collateral vessels, and these results demonstrate that satellite cells produce paracrine factors that increase both of these critical processes. To test the potential therapeutic capability of satellite cells in vivo, alginate encapsulated satellite cells were delivered in the hind limb at the time of the ischemia procedure. Alginate encapsulation has been previously shown to increase the cell retention and viability of other cell types, and using a whole animal in vivo imager to track luciferase expression of the cells, we found the encapsulated cells were viable for up to 2 weeks. The satellite cells also improved the recovery with the mice that received satellite cells having significantly increased perfusion over the mice that received empty capsules at 2 weeks post‐surgery as measured by Laser Doppler imaging (perfusion ratio of 0.87 ± 0.04 (cells) vs 0.68 ± 0.07 (empty capsules), p&lt;0.05). In conclusion our studies have shown that satellite cells proliferate in response to ischemia, produce paracrine factors that increase vascular cell migration in vitro, and lead to functional increases in perfusion in vivo. We believe these results demonstrate the critical role satellite cells play in collateral vessel formation and may be a potential new therapeutic approach for treating peripheral artery disease.Support or Funding InformationNIH F32HL124974 (LH)

Reduced perfusion in systemic sclerosis digital ulcers (both fingertip and extensor) can be increased by topical application of glyceryl trinitrate

ObjectivesIn patients with systemic sclerosis (SSc), fingertip digital ulcers (DUs) are believed to be ischaemic, and extensor surface DUs a result of mechanical factors/microtrauma. Our aim was to assess blood flow response to topical glyceryl trinitrate (GTN) compared to placebo in SSc DUs, looking for differences in pathophysiology between fingertip and extensor lesions. MethodThis was a double-blind, randomised, crossover, placebo-controlled study. Sixteen (6 fingertip, 10 extensor) DUs were each studied twice (one day apart): once with GTN and once with placebo ointment. Perfusion at the DU centre (‘DUCore’) and periphery (‘DUPeriphery’), as measured by laser Doppler imaging was performed before and immediately after ointment application, then every 10min, up to 90min post-application. We calculated the area under the response curve (AUC) and the ratio of peak perfusion to baseline, then compared these between GTN and placebo. ResultsPerfusion was lower in the DUCore compared to the DUPeriphery (ratio of 0.52). The microvessels of the DUCore were responsive to GTN, with an increase in perfusion, with a similar effect in both fingertip and extensor DUs. The AUC and peak/baseline perfusion difference in means (ratio, 95% confidence interval) between GTN and placebo at the DUCore were 1.2 (1.0–1.6) and 1.2 (1.0–1.5) respectively, and at the DUPeriphery were 1.1 (0.8–1.6) and 1.0 (0.9–1.2) respectively. ConclusionDUs (both fingertip and extensor) were responsive to topical GTN, with an increase in perfusion to the ischaemic DU centre. If both fingertip and extensor DUs have a (potentially reversible) ischaemic aetiology, this has important treatment implications.

Ponatinib Exerts Multiple Effects on Vascular Endothelial Cells: Possible Mechanisms and Explanations for the Adverse Vascular Events Seen in CML Patients Treated with Ponatinib

The potent BCR-ABL1-targeting tyrosine kinase inhibitor (TKI) ponatinib is used for the treatment of patients with drug-resistant chronic myeloid leukemia (CML). However, an increased risk of development of cardiovascular events has been described in CML patients treated with ponatinib. The etiology of these adverse events is currently unknown. In an attempt to discover mechanisms underlying ponatinib-induced adverse vascular events, we have evaluated the effects of ponatinib in vitro on human vascular endothelial cells and on contraction of explanted mice aortic rings. In addition, we examined the effects of ponatinib on angiogenesis in vivo in a mouse model of hind limb ischemia. Ponatinib dose-dependently induced apoptosis in human coronary artery endothelial cells (HCAEC) in a caspase assay (relative apoptosis vs. 1% DMSO: ponatinib 50 nM: 1.79±0.38, p<0.001; ponatinib 100 nM: 2.13±0.42, p<0.001) and this drug effect could partially be blocked by addition of insulin (ponatinib 100 nM + insulin 5 µg/ml: 1.70±0.18, p<0.05). In addition, ponatinib was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and the human microvascular endothelial cell line HMEC-1, with IC50 values ranging between 100 and 250 nM (p<0.05) as determined by thymidine-incorporation assay. Using a phospho-receptor tyrosine kinase assay in HCAEC, we found that ponatinib also inhibits fetal bovine serum-induced phosphorylation of the VEGF receptor KDR as well as phosphorylation of MER and insulin receptors, which play a role in angiogenesis, vascular homeostasis, and vessel protection. We also found that ponatinib (1 µM, 4 hours) increases adhesion of HUVEC to a plastic-surface compared to DMSO control (Figure). Based on clinical observations of vasoconstriction in ponatinib-treated patients, we also applied ponatinib on aortic rings harvested from C57BL/6 mice. Ponatinib (100 nM, overnight) enhanced norepinephrine-induced vasoconstriction (log EC50: control -7.76±0.06 vs. ponatinib -7.96±0.05, p<0.05, n=6) and inhibited acetylcholine-mediated vasodilatation (log IC50: control -7.45±0.05 vs. ponatinib -7.06±0.1, p<0.001) as shown by myography. These drug effects were blocked by inhibition of nitric oxide (using nitric oxide synthase inhibitor L-NNA, 100 µM) or COX (by applying diclofenac, 3 mg/l), suggesting that ponatinib promotes the generation of vasoconstricting prostanoids. Ponatinib effects were also blocked by the calcium channel blocker nifedipine (1 µM). In C57BL/6 mice, ponatinib (5 mg/kg/day for 35 days) was found to inhibit blood flow recovery in a hind limb ischemia model as shown by Laser-Doppler perfusion imaging after femoral artery ligation. The blood perfusion ratios of the ischemic limb vs. non-ischemic limb at week 5 were: control group: 0.67±0.07 vs. ponatinib: 0.56±0.1; p<0.05). Ponatinib-treated mice also developed toe and foot necrosis more frequently than control mice (necrosis score: control: 0.3 vs. ponatinib: 1.3). In summary, ponatinib affects endothelial cell growth and vasomotor function in-vitro as well as blood flow recovery in a mouse model. These findings might help explain the occurrence of vascular events in CML patients treated with ponatinib and may lead to development of therapeutic strategies for prevention and treatment of ponatinib-induced adverse events. [Display omitted] DisclosuresKirchmair:Ariad: Research Funding. Valent:Ariad: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Effect of cable cerclage on regional blood circulation in rabbits: a scintigraphic study.

To evaluate changes in blood circulation of the femoral cortex in rabbits using scintigraphy before and after cable cerclage alone or combined with an intramedullary Kirschner wire. Ten New Zealand rabbits were used. For the right femur, a 2-mm-thick cable was placed around the mid-diaphyseal region and squeezed with a 400-N force and locked with a clip. For the left femur, a 1.8-mm Kirschner wire was inserted retrogradely into the medullary canal, and a 2-mm-thick cable was applied using the same technique. The blood perfusion ratio of the region of interest (ROI) before and after surgery was evaluated using scintigraphy. For the right femurs, the mean ROI perfusion ratio decreased by 45% from 2.51 to 1.37 after intervention (p=0.001). For the left femurs, the mean ROI perfusion ratio decreased by 56% from 2.12 to 0.92 after intervention (p<0.001). The mean ROI perfusion ratio post-intervention was higher in the right than left femurs (p=0.017). Cable cerclage around the femoral cortex significantly decreased blood circulation in the area.