Functional Capillary Research Articles

Physical exercise promotes astrocyte coverage of microvessels in a model of chronic cerebral hypoperfusion

BackgroundBrain circulation disorders such as chronic cerebral hypoperfusion have been associated with a decline in cognitive function during the development of dementia. Astrocytes together with microglia participate in the immune response in the CNS and make them potential sentinels in the brain parenchyma. In addition, astrocytes coverage integrity has been related to brain homeostasis. Currently, physical exercise has been proposed as an effective intervention to promote brain function improvement. However, the neuroprotective effects of early physical exercise on the astrocyte communication with the microcirculation and the microglial activation in a chronic cerebral hypoperfusion model are still unclear. The aim of this study was to investigate the impact of early intervention with physical exercise on cognition, brain microcirculatory, and inflammatory parameters in an experimental model of chronic cerebral hypoperfusion induced by permanent bilateral occlusion of the common carotid arteries (2VO).MethodsWistar rats aged 12 weeks were randomly divided into four groups: Sham-sedentary group (Sham-Sed), Sham-exercised group (Sham-Ex), 2VO-sedentary group (2VO-Sed), and 2VO-exercised group (2VO-Ex). The early intervention with physical exercise started 3 days after 2VO or Sham surgery during 12 weeks. Then, the brain functional capillary density and endothelial-leukocyte interactions were evaluated by intravital microscopy; cognitive function was evaluated by open-field test; hippocampus postsynaptic density protein 95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the capillaries, microglial activation, and structural capillary density were evaluated by immunohistochemistry.ResultsEarly moderate physical exercise was able to normalize functional capillary density and reduce leukocyte rolling in the brain of animals with chronic cerebral hypoperfusion. These effects were accompanied by restore synaptic protein and the improvement of cognitive function. In addition, early moderate exercise improves astrocytes coverage in blood vessels of the cerebral cortex and hippocampus, decreases microglial activation in the hippocampus, and improves structural capillaries in the hippocampus.ConclusionsMicrocirculatory and inflammatory changes in the brain appear to be involved in triggering a cognitive decline in animals with chronic cerebral ischemia. Therefore, early intervention with physical exercise may represent a preventive approach to neurodegeneration caused by chronic cerebral hypoperfusion.

Impaired skeletal muscle performance as a consequence of random functional capillary rarefaction can be restored with overload-dependent angiogenesis.

Key points Loss of skeletal muscle capillaries is thought to contribute to a reduction in exercise tolerance, but the relative contribution of a compromised microcirculation with disease, in isolation of co‐morbidities, to impaired muscle function is unknown.We therefore developed a novel method to randomly occlude capillaries in the rat hindlimb to mimic the capillary rarefaction observed in many conditions.We demonstrate that muscle fatigue resistance is closely coupled with functional microvascular density, independent of arterial blood flow, while disturbance of the microcirculation leads to long‐term impairment of muscle function if left untreated.Mechanical stretch due to muscle overload causes a restoration of fatigue resistance via angiogenic remodelling.These observations highlight the importance of a healthy microcirculation and suggest that restoring impaired microvascular supply, regardless of disease co‐morbidities, will assist recovery of exercise tolerance in a variety of conditions that limit quality of life. To what extent microvascular rarefaction contributes to impaired skeletal muscle function remains unknown. Our understanding of whether pathological changes in the microcirculation can be reversed remains limited by a lack of basic physiological data in otherwise healthy tissue. The principal objectives here were to: (1) quantify the effect of random microvascular rarefaction on limb perfusion and muscle performance, and (2) determine if these changes could be reversed. We developed a novel protocol in rats whereby microspheres injected into the femoral artery allowed a unilateral reduction in functional capillary density in the extensor digitorum longus (EDL), and assessed acute and chronic effects on muscle function. Simultaneous bilateral EDL force and hindlimb blood flow measurements were made during electrical stimulation. Following functional capillary rarefaction there was an acute microsphere dose‐dependent reduction in muscle fatigue resistance (P < 0.001), despite preserved femoral artery perfusion. Histological analysis of EDL samples taken from injected animals confirmed a positive correlation between the proportion of functional capillaries and fatigue resistance (P = 0.002). Such impaired performance persisted for at least 2 weeks (P = 0.016). Concomitant mechanical overload improved both perfused capillary density and fatigue resistance (P<0.05), confirming that the capacity for muscle remodelling was retained following chronic distributed ischaemia, and that the impact of capillary rarefaction could be alleviated. These results demonstrate that loss of functional capillaries is detrimental to muscle function, even in otherwise healthy tissue, independent of arterial perfusion. Restoration of muscle performance following a mechanical overload stimulus indicates that angiogenic treatments to alleviate microvascular rarefaction may be key to restoring exercise tolerance.

A Model of Indirect Cell Death Caused by Tumor Vascular Damage after High-Dose Radiotherapy.

There is increasing evidence that high doses of radiotherapy, like those delivered in stereotactic body radiotherapy (SBRT), trigger indirect mechanisms of cell death. Such effect seems to be two-fold. High doses may trigger an immune response and may cause vascular damage, leading to cell starvation and death. Development of mathematical response models, including indirect death, may help clinicians to design SBRT optimal schedules. Despite increasing experimental literature on indirect tumor cell death caused by vascular damage, efforts on modeling this effect have been limited. In this work, we present a biomathematical model of this effect. In our model, tumor oxygenation is obtained by solving the reaction-diffusion equation; radiotherapy kills tumor cells according to the linear-quadratic model, and also endothelial cells (EC), which can trigger loss of functionality of capillaries. Capillary death will affect tumor oxygenation, driving nearby tumor cells into severe hypoxia. Capillaries can recover functionality due to EC proliferation. Tumor cells entering a predetermined severe hypoxia status die according to a hypoxia-death model. This model fits recently published experimental data showing the effect of vascular damage on surviving fractions. It fits surviving fraction curves and qualitatively reproduces experimental values of percentages of functional capillaries 48 hours postirradiation, and hypoxic cells pre- and 48 hours postirradiation. This model is useful for exploring aspects of tumor and EC response to radiotherapy and constitutes a stepping stone toward modeling indirect tumor cell death caused by vascular damage and accounting for this effect during SBRT planning. SIGNIFICANCE: A novel biomathematical model of indirect tumor cell death caused by vascular radiation damage could potentially help clinicians interpret experimental data and design better radiotherapy schedules.

Sedation with midazolam worsens the diaphragm function than dexmedetomidine and propofol during mechanical ventilation in rats

BackgroundMechanical ventilation (MV) is identified as an independent contributor to diaphragmatic atrophy and contractile dysfunction. Appropriate sedation is also essential during MV, and anesthetics may have direct adverse effects on the diaphragm. However, there is a lack of research into the effects of different anesthetics on diaphragm function during MV. ObjectivesIn the present study, we aim to examine the effect of midazolam, dexmedetomidine, and propofol on diaphragm function during MV. DesignAnimal study. SettingUniversity research laboratory. SubjectsMale Wistar rats. InterventionsAnimals were experienced 12 h of MV or spontaneous breathing (SB) with continuous anesthetics infusion. Diaphragm contractile properties, cross-sectional areas, microcirculation, oxidative stress, and proteolysis were examined. Measurements and main resultsDiaphragmatic specific force was markedly reduced in the midazolam group compared with the dexmedetomidine (−60.4 ± 3.01%, p < 0.001) and propofol group (−58.3 ± 2.60%, p < 0.001) after MV. MV sedated with midazolam induced more atrophy of type II fibers compared with dexmedetomidine (−21.8 ± 2.11%, p = 0.0001) and propofol (−8.2 ± 1.53%, p = 0.003). No significant differences of these indices were found in the midazolam, dexmedetomidine, and propofol groups under SB condition (all p > 0.05, respectively). Twelve hours of MV resulted in a time dependent reduction in diaphragmatic functional capillary density (PB −25.1%, p = 0.0001; MZ −21.6%, p = 0.0003; DD −15.2%, p = 0.022; PP −24.8%, p = 0.0001, respectively), which did not occur in the gastrocnemius muscle. The diaphragmatic lipid peroxidation adducts 4-HNE and HIF-1α levels were significantly lower in dexmedetomidine group and propofol group compared to midazolam group (p < 0.05, respectively). Meanwhile, the catalase and SOD levels were also relatively lower (p < 0.05, respectively) in midazolam group compared to dexmedetomidine group and propofol group. ConclusionsTwelve hours of mechanical ventilation during midazolam sedation led to a more severe diaphragm dysfunction than dexmedetomidine and propofol, possibly caused by its relative weaker antioxidant capacity.

Dexmedetomidine as an Alternative Anesthetic Agent for Flap Surgery: An Intravital Evaluation in the Cremaster Muscle Flap

Flap surgery is one of the most commonly used techniques of reconstructive surgery for effective repair of damaged tissue. Optimal anesthetic technique and anesthetic agent plays an important role in flap perfusion. This study aimed to evaluate the effects of dexmedetomidine infusion on the microcirculation in the cremaster muscle flap by direct in vivo monitoring. We randomly divided 9 Wistar albino rats into 3 groups. The rats in the control group underwent the surgical procedure (isolation of the cremaster muscle) alone; the rats in the experimental groups 1 and 2 received an infusion of dexmedetomidine (10 and 30 min) after the surgical procedure. The means of vessel diameters, number of functional capillaries, and movements of leukocytes in all groups were evaluated using intravital microscopic examination. The diameters of the arterioles and venules of the cremaster muscle significantly increased in the dexmedetomidine groups. The number of functional capillaries was higher in the dexmedetomidine groups than in the control group. No difference was observed in the movements of leukocytes between the control and experimental groups. Dexmedetomidine significantly increased the diameters of the arterioles and venules of the cremaster flap and the number of functional capillaries. On the basis of the effects of dexmedetomidine on microcirculation, we suggest that dexmedetomidine continue to be used as an anesthetic agent, and may be considered also for reconstructive procedures, particularly flap surgery.

Local Biological Reactions and Pseudotumor-Like Tissue Formation in relation to Metal Wear in a Murine In Vivo Model.

Metal wear debris and released ions (CoCrMo), which are widely generated in metal-on-metal bearings of hip implants, are also found in patients with metal-on-polyethylene bearings due to the mechanically assisted crevice corrosion of modular taper junctions, including head-neck and neck-stem taper interfaces. The resulting adverse reactions to metal debris and metal ions frequently lead to early arthroplasty revision surgery. National guidelines have since been published where the blood metal ion concentration of patients must consistently be monitored after joint replacement to prevent serious complications from developing after surgery. However, to date, the effect of metal particles and metal ions on local biological reactions is complex and still not understood in detail; the present study sought to elucidate the complex mechanism of metal wear-associated inflammation reactions. The knee joints in 4 groups each consisting of 10 female BALB/c mice received injections with cobalt chrome ions, cobalt chrome particles, and ultra-high-molecular-weight polyethylene (UHMWPE) particles or PBS (control). Seven days after injection, the synovial microcirculation and knee joint diameter were assessed via intravital fluorescence microscopy followed by histological evaluation of the synovial layer. Enlarged knee diameter, enhanced leukocyte to endothelial cell interactions, and an increase in functional capillary density within cobalt chrome particle-treated animals were significantly greater than those in the other treatment groups. Subsequently, pseudotumor-like tissue formations were observed only in the synovial tissue layer of the cobalt chrome particle-treated animals. Therefore, these findings strongly suggest that the cobalt chrome particles and not metal ions are the cause for in vivo postsurgery implantation inflammation.

High-affinity Cu(I) chelator PSP-2 as potential anti-angiogenic agent

Copper is an essential trace metal that has been implicated in angiogenesis, the formation of new blood vessels. As tumor growth relies on establishing a functional capillary network for blood supply, copper chelation therapy may hold promise as an anti-cancer strategy by suppressing angiogenesis. To test the anti-angiogenic effect of PSP-2, a recently developed high affinity Cu(I) chelator with low zeptomolar dissociation constant, we utilized the endothelial cancer cell line EAhy926 and assessed changes in cell migration, proliferation, and tube formation in Matrigel. In addition, sprouting was assessed by the chicken and sheep aortic ring assay, and vascular pattern formation was studied in the chorioallantoic membrane of chicken embryos (CAM assay). While incubation with PSP-2 resulted in selective depletion of cellular copper levels, cell migration was not affected and the proliferating activity was even slightly increased. Moreover, the endothelial tube formation assay revealed significant morphological changes in the presence of PSP-2, with thicker tubular walls and an overall decreased meshes area. Similarly, the aortic ring assay and CAM assay showed that PSP-2 evokes significantly longer sprouts with smaller angles at branching points. Altogether, PSP-2 exhibits significant bioactivity at concentrations as low as 5 μM, rendering it a promising anti-angiogenic agent. As EAhy926 cells exhibit both endothelial and tumorigenic properties, the anti-angiogenic effect of PSP-2 might potentially translate also into anti-cancer activity.

Reperfusion Microvascular Ischemia After Prolonged Coronary Occlusion: Implications And Treatment With Local Supersaturated Oxygen Delivery.

Following a prolonged coronary arterial occlusion, heterogeneously scattered, focal regions of low erythrocyte flow are commonly found throughout the reperfused myocardium. Experimental studies have also demonstrated the presence of widespread, focally patchy regions of microvascular ischemia during reperfusion (RMI). However, the potential contribution of RMI to tissue viability and function has received little attention in the absence of practical clinical methods for its detection. In this review, the anatomic/functional basis of RMI is summarized, along with the evidence for its presence in reperfused myocardium. Advances in microcirculation research related to obstructive responses of vascular endothelial cells and blood elements to the effects of hypoxia and low shear stress are discussed, and a potential cycle of intensification of RMI from such responses and progressive loss of functional capillary density is presented. In capillaries with impaired erythrocyte flow, compensatory increases in the delivery of oxygen, because of its low solubility in plasma, are effective only at high partial pressures. As discussed herein, attenuation of the cycle with oxygen at hyperbaric levels in plasma is, very likely, responsible for improved tissue level perfusion noted experimentally. Observed clinical benefits from intracoronary SuperSaturated oxygen (SSO2) delivery, including infarct size reduction, can be attributed to attenuation of RMI with improvement in microvascular blood flow.

Fluid management in the perioperative setting: mind the kidney

Acute kidney injury (AKI) is one of the most frequent complications in critically ill patients and in the perioperative setting. The anatomical structure and the microvasculature of the kidney makes it highly vulnerable to hypoxia. Although fluid therapy is considered crucial in situations where improvement of cardiac output is needed, it can also contribute to AKI development when administered inappropriately. Hemodilution and anemia during cardio-pulmonary bypass have been demonstrated to be risk factors for AKI and they are likely to be a consequence of fluid administration. In order to assess the perfusion of the kidneys it is necessary to investigate the determinants of delivery of oxygen at the microcirculatory level. Indeed, fluids can decrease the capillary hematocrit and the functional capillary density, affecting the renal oxygenation and increasing the risk of AKI. Monitoring sublingual microcirculation can be a reliable tool to guide fluid administration, aiming to prevent or improve perioperative AKI.

Microcirculation and haemodynamics after infraclavicular brachial plexus block using adrenaline as an adjuvant to lidocaine: a randomised, double-blind, crossover study inhealthy volunteers.

We evaluated the effect of adrenaline on human skin microcirculation (nutritive and sub-papillary) and systemic cardiovascular variables after it was added to lidocaine in infraclavicular brachial plexus blocks. Twelve healthy, non-smoking male volunteers were included, each attending two study sessions 2weeks apart, and they were studied using a crossover design. In both sessions, they received an ultrasound-guided infraclavicular brachial plexus block in the non-dominant arm with 0.4ml.kg-1 lidocaine, 15mg.ml-1 with or without adrenaline 5μg.ml-1 . Microcirculation was assessed by laser Doppler fluxmetry (sub-papillary blood flow), capillary video microscopy (nutritive blood flow) and continuous temperature measurements. Heart rate and arterial pressure were recorded continuously and non-invasively. Median (IQR [range]) sub-papillary blood flow increased substantially 30min after the brachial plexus block, from 8.5 (4.4-13.5 [2.9-28.2]) to 162.7 (111.0-197.8 [9.5-206.7]) arbitrary units with adrenaline (p=0.017), and from 6.9 (5.3-28.5 [1.8-42.1] to 133.7 (16.5-216.7 [1.0-445.0] arbitrary units without adrenaline (p=0.036). Nutritive blood flow (functional capillary density, capillaries.mm-2 , measured at the dorsal side of the hand) decreased in the blocked extremity when adrenaline was used as adjuvant, from median (IQR [range]) 45 (36-52 [26-59]) to 38 (29-41 [26-42]), p=0.028, whereas no significant change occurred without adrenaline. Median finger skin temperature (°C) increased by 44% (data pooled) with no significant differences between the groups. No significant changes were found in the systemic cardiovascular variables with or without adrenaline. We conclude that lidocaine infraclavicular brachial plexus blocks caused an increase in skin sub-papillary blood flow. The addition of adrenaline produced stronger and longer lasting blocks, but decreased the nutritive blood flow.

Dissociation between macro- and microvascular parameters in the early phase of hemorrhagic shock

Hemorrhagic shock (HS) therapy is based on macrohemodynamic improvement, but it is not clear if this therapy correlates directly with increases in tissue perfusion. Aiming to clarify this point, we compared norepinephrine (NE, a vasoconstrictor), sodium nitroprusside (NP, a vasodilator) and levosimendan (LEV, an inodilator) treatments on macro and microvascular parameters using the hamster dorsal skinfold chamber preparation. One hour after HS, animals received Ringer's lactate (RL) solution within 10 min, then animals received RL, NP, NE and LEV during 90 min via jugular vein. Macrovascular variables: mean arterial pressure (MAP), heart rate (HR), maximal ventricle pressure (MVP), change in ventricular pressure over time (dP/dt) and microvascular variables: arteriolar and venular diameters, functional capillary density (FCD) and red blood cell velocity (RBCV) were evaluated at baseline, 60 min after HS, 60 and 90 min after treatments. Lactate blood concentrations were evaluated at baseline, 60 min after HS and 90 min after treatments. Hematocrit (Hct), cardiac output (CO), stroke volume (SV) and number of rolling and adhered leukocytes were assessed at 90 min after treatments. Data were considered significant when p < 0.05. NE increased significantly all macrohemodynamic variables compared to baseline (except MAP), and it was the only treatment that increased Hct, CO and SV significantly. NE decreased significantly all microvascular variables in comparison to baseline. NP increased HR, FCD and RBCV and reduced MVP and dP/dt significantly. LEV decreased MVP and dP/dt, arteriolar diameter and FCD and augmented RBCV significantly in comparison to baseline. Blood concentration of lactate increased significantly 60 min after HS. Leukocyte rolling and adhesion were not different between groups. We concluded that, early, during hemorrhagic shock, norepinephrine associated to fluid therapy improved macrohemodynamic parameters but failed to improved microvascular flow. Conversely, sodium nitroprusside association had the opposite effect. Despite its inodilator properties, levosimendan did not improve macro or microhemodynamic parameters when combined to fluid therapy.

Pulmonary capillary surface area in supine exercising humans: demonstration of vascular recruitment.

In exercising humans, cardiac output (CO) increases, with minor increases in pulmonary artery pressure (PAP). It is unknown if the CO is accommodated via distention of already perfused capillaries or via recruitment of nonconcomitantly perfused pulmonary capillaries. Ten subjects (9 female) performed symptom-limited exercise. Six had resting mean PAP (PAPm) <20 mmHg, and four had PAPm between 21 and 24 mmHg. The first-pass pulmonary circulatory metabolism of [3H]benzoyl-Phe-Ala-Pro (BPAP) was measured at rest and at peak exercise, and functional capillary surface area (FCSA) was calculated. Data are means ± SD. Mean pulmonary arterial pressure rose from 18.8 ± 3.3 SD mmHg to 28.5 ± 4.6 SD mmHg, CO from 6.4 ± 1.6 to 13.4 ± 2.9 L/min, and pulmonary artery wedge pressure from 14 ± 3.3 to 19.5 ± 5 mmHg (all P ≤ 0.001). Percent BPAP metabolism fell from 74.7 ± 0.1% to 67.1 ± 0.1%, and FCSA/body surface area (BSA) rose from 2,939 ± 640 to 5,018 ± 1,032 mL·min-1·m-2 (all P < 0.001). In nine subjects, the FCSA/BSA-to-CO relationship suggested principally capillary recruitment and not distention. In subject 10, a marathon runner, resting CO and FCSA/BSA were high, and increases with exercise suggested distention. Exercising humans demonstrate pulmonary capillary recruitment and distention. At moderate resting CO, increasing blood flow causes principally recruitment while, based on one subject, when exercise begins at high CO, further increases appear to cause distention. Our findings clarify an important physiologic question. The technique may provide a means for further understanding exercise physiology, its limitation in pulmonary hypertension, and responses to therapy.

Nerve/glial antigen 2 is crucially involved in the revascularization of freely transplanted pancreatic islets

Pancreatic islets are highly vascularized endocrine units. Accordingly, their adequate revascularization is of major importance for successful islet transplantation. The proteoglycan, nerve/glial antigen 2 (NG2) expressed in pericytes is a crucial regulator of angiogenesis. Therefore, we herein analyze whether this surface protein contributes to the revascularization of grafted islets. Islets were isolated from NG2+/+ (wild-type) and NG2-/- mice and their cellular composition was analyzed by immunohistochemical detection of insulin, glucagon, somatostatin and CD31. Moreover, insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). In addition, isolated islets were transplanted into dorsal skinfold chambers of wild-type mice and their revascularization was determined by intravital fluorescence microscopy and immunohistochemistry. NG2+/+ and NG2-/- islets did not differ in their cellular composition and insulin secretion. However, transplanted NG2-/- islets exhibited a significantly lower functional capillary density and a reduced number of CD31-positive microvessels. These findings demonstrate that the loss of NG2 impairs the revascularization of transplanted islets, underlining the importance of this pericytic proteoglycan for islet engraftment.

Reducing Ischemia/Reperfusion Injury by the Targeted Delivery of Nitric Oxide from Magnetic-Field-Induced Localization of S-Nitrosothiol-Coated Paramagnetic Nanoparticles.

Nitric oxide (NO) is an important factor during an ischemia/reperfusion (I/R) injury. Protective actions of NO during I/R are attributed to antioxidant and anti-inflammatory effects, as well as cell-signaling-based inhibition of nuclear proteins. The therapeutic potential of supplemented NO during I/R is nonetheless uncertain, since peroxynitrite formed from NO near superoxide can be potentially harmful due to NF-κB up-regulation and direct cytotoxicity. This study investigates new technology to provide the magnet-assisted delivery of therapeutic levels of localized NO to targeted I/R tissues using biocompatible gadolinium-oxide-based paramagnetic nanoparticles coated with S-nitrosothiols (SNO-PMNPs). Hamsters fitted with a window chamber were subjected to ischemia by application of a tourniquet at the periphery of the window chamber for 1 h. The SNO-PMNPs were intravenously infused (10 mg/kg) during the reperfusion phase, during which time a localized external magnetic field was either applied or not applied to the I/R area. The microvascular hemodynamics, functional capillary density (FCD), rolling and adherent leukocytes, reactive oxygen and nitrogen species, and tissue viability were assessed using intravital microscopy. Control animals did not receive SNO-PMNPs. Treatment with SNO-PMNPs plus a magnet but not without a magnet increased reflow, decreased leukocytes rolling and sticking in postcapillary venules, limited cell death, and restored the FCD. The absence of the magnet resulted in systemic changes in heart rate and mean arterial blood pressure, consistent with the systemic delivery of NO by the SNO-PMNP. These results indicate that the localized delivery of NO during reperfusion counters the deleterious consequences of peroxynitrite and other reactive species generated upon reperfusion as reflected in localized increases in blood flow and tissue viability, all with minimal systemic effects. This technology can provide the basis for a timely treatment of a localized ischemia-associated disease to prevent injury in different tissues and organs.

Imaging the injured beating heart intravitally and the vasculoprotection afforded by haematopoietic stem cells

AimsAdequate microcirculatory perfusion, and not just opening of occluded arteries, is critical to salvage heart tissue following myocardial infarction. However, the degree of microvascular perfusion taking place is not known, limited primarily by an inability to directly image coronary microcirculation in a beating heart in vivo. Haematopoietic stem/progenitor cells (HSPCs) offer a potential therapy but little is known about their homing dynamics at a cellular level and whether they protect coronary microvessels. This study used intravital microscopy to image the anaesthetized mouse beating heart microcirculation following stabilization.Methods and resultsA 3D-printed stabilizer was attached to the ischaemia–reperfusion injured (IRI) beating heart. The kinetics of neutrophil, platelet and HSPC recruitment, as well as functional capillary density (FCD), was imaged post-reperfusion. Laser speckle contrast imaging (LSCI) was used for the first time to monitor ventricular blood flow in beating hearts. Sustained hyperaemic responses were measured throughout reperfusion, initially indicating adequate flow resumption. Intravital microscopy confirmed large vessel perfusion but demonstrated poor transmission of flow to downstream coronary microvessels. Significant neutrophil adhesion and microthrombus formation occurred within capillaries with the latter occluding them, resulting in patchy perfusion and reduced FCD. Interestingly, ‘patrolling’ neutrophils were also observed in capillaries. Haematopoietic stem/progenitor cells readily trafficked through the heart but local retention was poor. Despite this, remarkable anti-thromboinflammatory effects were observed, consequently improving microvascular perfusion.ConclusionWe present a novel approach for imaging multiple microcirculatory perturbations in the beating heart with LSCI assessment of blood flow. Despite deceptive hyperaemic responses, increased microcirculatory flow heterogeneity was seen, with non-perfused areas interspersed with perfused areas. Microthrombi, rather than neutrophils, appeared to be the major causative factor. We further applied this technique to demonstrate local stem cell presence is not a pre-requisite to confer vasculoprotection. This is the first detailed in vivo characterization of coronary microcirculatory responses post-reperfusion injury.

Organ dysfunction in cirrhosis: a mechanism involving the microcirculation.

Microcirculation is essential for adequate tissue perfusion and organ function. Microcirculatory changes may occur in cirrhosis, inducing loss of multiorgan function. The aim was to evaluate preliver transplantation and postliver transplantation aspects of multiorgan function, microcirculation, inflammatory, and endothelial biomarkers and survival in a controlled study including cirrhotic outpatients. We accessed functional capillary density (FCD) and red blood cell acceleration (RBCA) by nailfold videocapillaroscopy. Inflammatory and endothelial biomarkers [interleukin-6 (IL-6), soluble intercellular adhesion molecule-1, endothelin-1, and tumor necrosis factor-α] were analyzed. Cerebral and renal functions were assessed to represent organ dysfunction and regression analyses were carried out. Receiver operating characteristic curves were constructed and survival Kaplan-Meier analysis was carried out. Fifty-four patients and 18 controls were included. Inflammatory and endothelial markers increased in advanced disease. FCD was reduced and RBCA was progressively lower according to disease severity. RBCA correlated inversely with inflammatory and endothelial biomarkers, and directly with renal function. The presence of hepatic encephalopathy correlated inversely with RBCA and directly with IL-6 and endothelin-1. In multivariate analysis, RBCA was an independent factor for organ dysfunction. The area under the receiver operating chartacteristic curve for IL-6 for survival was 0.74 (0.59-0.89), P=0.05. Transplant-free survival was 97.5% for values under 5.78 ng/ml (IL-6 best cutoff) and 83.9% above 5.78 ng/ml, log-rank=0.018. Eleven patients underwent transplantation, with an overall improvement in microcirculatory function. Our results suggest a mechanism of organ damage in cirrhosis, where microcirculatory dysfunction could be correlated to inflammatory and endothelial biomarkers, and loss of multiorgan function. IL-6 seems to be an important survival marker of inflammation. Liver transplantation improved microcirculatory dysfunction, corroborating this hypothesis.

Functional capillary impairment in patients with ventricular assist devices

The implantation of continuous – flow ventricular assist devices (VAD) is suggested to evoke angiodysplasia contributing to adverse events such as gastrointestinal bleeding. We evaluated in vivo capillary density and glycocalyx dimensions to investigate possible systemic microvascular changes in patients with chronic heart failure and VAD support vs. standard medical treatment. Forty-two patients with VAD support were compared to forty-one patients with ischemic and non-ischemic chronic heart failure (CHF) on standard pharmacotherapy and to a group of forty-two healthy subjects in a prospective cross-sectional study. Sublingual microcirculation was visualized using Sidestream Darkfield videomicroscopy and functional and perfused total capillary densities were quantified. Patients with VAD implantation were followed for one year and bleeding events were recorded. Median time after VAD implantation was 18 months. Patients were treated with centrifugal-flow devices (n = 31) or axial-flow devices (n = 11). Median functional capillary density was significantly lower in patients with VAD therapy as compared to CHF patients (196 vs. 255/mm2, p = 0.042, adjusted p-value). Functional and total capillary densities were 44% and 53% lower (both p < 0.001) in patients with VAD therapy when compared to healthy subjects. Cox regression analysis revealed loss of capillary density as a significant predictor of bleeding events during one -year follow-up of VAD patients (HR: 0.987, CI (95%): 0.977–0.998, p = 0.021 for functional and 0.992, CI (95%): 0.985–0.999, p = 0.03 for total capillary density). In conclusion, patients with VAD support exhibit capillary density rarefaction, which was associated with bleeding events. If confirmed independently, capillary impairment may be evaluated as novel marker of bleeding risk.

Alterations in myocardial connexin-43 and matrix metalloproteinase-2 signaling in response to pregnancy and oxygen deprivation of Wistar rats: a pilot study 1.

Two important aspects of cardiac adaptive response to pregnancy have been studied in normal as well as hypoxic conditions: (1) intercellular signaling mediated by myocardial connexin-43 (Cx43) that is crucial to synchronize heart function; (2) extracellular signaling mediated by matrix metalloproteinase-2 (MMP-2) that is an early marker of extracellular matrix remodeling. Myocardial Cx43 distribution and functional capillary density were determined as well. Hypoxia was induced by exposure of rats to 10.5% O2 and 89.5% N2 in a hermetically sealed chamber. Findings showed that pregnancy resulted in a significant increase of Cx43 protein expression, its functional phosphorylated forms, and enhanced capillary density while did not affect either expression of total MMP-2 or its activity. Maternal hypoxia for 12 or 16 h did not affect elevated Cx43 but enhanced its distribution on lateral sides of the cardiomyocytes. In contrast, hypoxia of nonpregnant rats resulted in upregulation of Cx43, its lateral distribution, and enhanced capillary density. Hypoxia did not affect myocardial MMP-2 either in pregnant or nonpregnant rats. Cardiac adaptive response to pregnancy is accompanied by enhanced Cx43 without changes in MMP-2 signaling. Pregnant rat heart is tolerant to short-term hypoxemia, while nonpregnant rat heart reacts by upregulation of Cx43 and increased capillary density.

Early Life Microcirculatory Plasticity and Blood Pressure Changes in Low Birth Weight Infants Born to Normotensive Mothers: A Cohort Study.

BACKGROUNDCapillary rarefaction (CR) is an established hallmark of essential hypertension (EH). The aim of this study was to examine early changes in capillary density (CD) and blood pressure (BP) in low birth weight (LBW) infants who are at risk of developing EH in later life.METHODSWe studied 77 LBW infants and 284 normal birth weight (NBW) infants, all born to mothers with normotension, in a longitudinal multicenter study. Intravital capillaroscopy was used to measure functional basal capillary density (BCD) and maximal capillary density (MCD) at birth, 3, 6, and 12 months.RESULTSWe found that LBW infants, born preterm and at term, had a significantly higher CD at birth, then underwent significant CR in the 1st 3 months culminating in a CD similar to that seen in NBW infants. NBW infants showed a gradual reduction in CD between birth and 12 months. Non-Caucasian ethnicity and preterm birth were significant predictors of a higher CD at birth. Systolic BP in NBW infants increased significantly from birth to 3 months, and we identified a significant negative correlation between systolic BP and MCD.CONCLUSIONSThis study has identified a process of early “accelerated capillary remodeling” in LBW infants, which corrects their higher CD at birth. This remodeling is unlikely to explain the CR seen in adult individuals with, or at risk of developing EH. Further follow-up studies are required to determine the timing and mechanisms involved in CR, which is likely to occur after the 1st year of life but before early adulthood.

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels.

The development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.