Hirotsugu Tsuchimochi

Synchrotron Radiation Imaging for Advancing Our Understanding of Cardiovascular Function

Synchrotron radiation (SR) is increasingly being used for micro-level and nano-level functional imaging in in vivo animal experiments. This review focuses on the methodology that enables repeated and regional assessment of vessel internal diameter and flow in the resistance vessels of different organ systems. In particular, SR absorption microangiography approaches offer unique opportunities for real-time in vivo vascular imaging in small animals, even during dynamic motion of the heart and lungs. We also describe recent progress in the translation of multiple phase-contrast imaging techniques from ex vivo to in vivo small-animal studies. Furthermore, we also review the utility of SR for multiple pinpoint (dimensions 0.2×0.2 mm) assessments of myocardial function at the cross-bridge level in different regions of the heart using small-angle X-ray scattering, resulting from increases in SR flux at modern facilities. Finally, we present cases for the use of complementary SR approaches to study cardiovascular function, particularly the pathological changes associated with disease using small-animal models.

Changes in functional and histological distributions of nitric oxide synthase caused by chronic hypoxia in rat small pulmonary arteries

Chronic hypoxia (CH) increases lung tissue expression of all types of nitric oxide synthase (NOS) in the rat. However, it remains unknown whether CH‐induced changes in functional and histological NOS distributions are correlated in rat small pulmonary arteries. We measured the effects of NOS inhibitors on the internal diameters (ID) of muscular (MPA) and elastic (EPA) pulmonary arteries (100–700 μm ID) using an X‐ray television system on anaesthetized rats. We also conducted NOS immunohistochemical localization on the same vessels. Nonselective NOS inhibitors induced ID reductions in almost all MPA of CH rats (mean reduction, 36±3%), as compared to ∼60% of control rat MPA (mean, 10±2%). The inhibitors reduced the ID of almost all EPA with similar mean values (∼26%) in both CH and control rats. On the other hand, inducible NOS (iNOS)‐selective inhibitors caused ID reductions in ∼60% of CH rat MPA (mean, 15±3%), but did so in only ∼20% of control rat MPA (mean, 2±2%). This inhibition caused only a small reduction (mean, ∼4%) in both CH and control rat EPA. A neuronal NOS‐selective inhibitor had no effect. The percentage of endothelial NOS (eNOS)‐positive vessels was ∼96% in both MPA and EPA from CH rats, whereas it was 51 and 91% in control MPA and EPA, respectively. The percentage for iNOS was ∼60% in both MPA and EPA from CH rats, but was only ∼8% in both arteries from control rats. The data indicate that in CH rats, both functional and histological upregulation of eNOS extensively occurs within MPA. iNOS protein increases sporadically among parallel‐arranged branches in both MPA and EPA, but its vasodilatory effect is predominantly observed in MPA. Such NOS upregulation may serve to attenuate hypoxic vasoconstriction, which occurs primarily in MPA and inhibit the progress of pulmonary hypertension.

Synchrotron-based angiography for investigation of the regulation of vasomotor function in the microcirculation in vivo.

1 Real‐time imaging of the vascular networks of any organ system in vivo is possible with synchrotron radiation (SR) angiography. In this review, we discuss the advantages of SR angiography over clinical X‐ray imaging and other non‐ionizing imaging modalities. Current limitations are also described. 2 The usefulness of dual‐energy and temporal subtraction approaches to K‐edge iodine imaging are compared. 3 High‐resolution images of the microcirculation in small animals are now being collected routinely by multiple research groups through public access research programmes at synchrotrons worldwide. Such images are permitting unrivalled insights into vasomotor regulation deep within intact organ systems, such as the brain, kidney, lung and heart. For example, recent observations indicate changes in vascular control mechanisms in pulmonary hypertension that are specific to certain branching segments of the pulmonary circulation. 4 New possibilities for non‐iodinated contrast agents in SR angiography are briefly described. 5 High‐resolution angiography in vivo using SR will now allow us to identify vessels with localized or non‐uniform vasoconstriction in states such as diabetes or to characterize the extent of endothelial dysfunction in the circulation following hypertension or ischaemic–reperfusion injury. In the near future, this research is expected to reveal the contribution of resistance vessel dysfunction to diverse pathophysiological states, such as stroke, hypertension and ischaemic heart disease.

APJ Regulates Parallel Alignment of Arteries and Veins in the Skin

Molecular pathways regulating the development of arterial and venous endothelial cells (ECs) are now well established, but control of parallel arterial-venous alignment is unclear. Here we report that arterial-venous alignment in the skin is determined by apelin receptor (APJ) expression in venous ECs. One of the activators of APJ is apelin. We found that apelin is produced by arterial ECs during embryogenesis, induces chemotaxis of venous ECs, and promotes the production of secreted Frizzled-related protein 1 (sFRP1) by APJ(+) ECs. sFRP1 stimulates matrix metalloproteinase production by Ly6B.2(+) neutrophil-like cells located between the arteries and veins, resulting in remodeling of extracellular matrices to support venous displacement. Moreover, using apelin- or APJ-deficient mice, which exhibit arterial-venous disorganization, we found that arterial-venous alignment is involved in thermoregulation, possibly by regulating countercurrent heat exchange. We hypothesize that the evolution of parallel juxtapositional arterial-venous alignment was an adaptation to reduce body heat loss.

Contractile apparatus dysfunction early in the pathophysiology of diabetic cardiomyopathy.

Diabetes mellitus significantly increases the risk of cardiovascular disease and heart failure in patients. Independent of hypertension and coronary artery disease, diabetes is associated with a specific cardiomyopathy, known as diabetic cardiomyopathy (DCM). Four decades of research in experimental animal models and advances in clinical imaging techniques suggest that DCM is a progressive disease, beginning early after the onset of type 1 and type 2 diabetes, ahead of left ventricular remodeling and overt diastolic dysfunction. Although the molecular pathogenesis of early DCM still remains largely unclear, activation of protein kinase C appears to be central in driving the oxidative stress dependent and independent pathways in the development of contractile dysfunction. Multiple subcellular alterations to the cardiomyocyte are now being highlighted as critical events in the early changes to the rate of force development, relaxation and stability under pathophysiological stresses. These changes include perturbed calcium handling, suppressed activity of aerobic energy producing enzymes, altered transcriptional and posttranslational modification of membrane and sarcomeric cytoskeletal proteins, reduced actin-myosin cross-bridge cycling and dynamics, and changed myofilament calcium sensitivity. In this review, we will present and discuss novel aspects of the molecular pathogenesis of early DCM, with a special focus on the sarcomeric contractile apparatus.

Cell-sheet Therapy With Omentopexy Promotes Arteriogenesis and Improves Coronary Circulation Physiology in Failing Heart

Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufficient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.

Functional and Electrical Integration of Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Myocardial Infarction Rat Heart.

In vitro expanded beating cardiac myocytes derived from induced pluripotent stem cells (iPSC-CMs) are a promising source of therapy for cardiac regeneration. Meanwhile, the cell sheet method has been shown to potentially maximize survival, functionality, and integration of the transplanted cells into the heart. It is thus hypothesized that transplanted iPSC-CMs in a cell sheet manner may contribute to functional recovery via direct mechanical effects on the myocardial infarction (MI) heart. F344/NJcl-rnu/rnu rats were left coronary artery ligated (n = 30), followed by transplantation of Dsred-labeled iPSC-CM cell sheets of murine origin over the infarct heart surface. Effects of the treatment were assessed, including in vivo molecular/cellular evaluations using a synchrotron radiation scattering technique. Ejection fraction and activation recovery interval were significantly greater from day 3 onward after iPSC-CM transplantation compared to those after sham operation. A number of transplanted iPSC-CMs were present on the heart surface expressing cardiac myosin or connexin 43 over 2 weeks, assessed by immunoconfocal microscopy, while mitochondria in the transplanted iPSC-CMs gradually showed mature structure as assessed by electron microscopy. Of note, X-ray diffraction identified 1,0 and 1,1 equatorial reflections attributable to myosin and actin–myosin lattice planes typical of organized cardiac muscle fibers within the transplanted cell sheets at 4 weeks, suggesting cyclic systolic myosin mass transfer to actin filaments in the transplanted iPSC-CMs. Transplantation of iPSC-CM cell sheets into the heart yielded functional and electrical recovery with cyclic contraction of transplanted cells in the rat MI heart, indicating that this strategy may be a promising cardiac muscle replacement therapy.

In Situ Measurements of Crossbridge Dynamics and Lattice Spacing in Rat Hearts by X-Ray Diffraction Sensitivity to Regional Ischemia

Background—Synchrotron radiation has been used to analyze crossbridge dynamics in isolated papillary muscle and excised perfused hearts with the use of x-ray diffraction techniques. We showed that these techniques can detect regional changes in rat left ventricle contractility and myosin lattice spacing in in situ ejecting hearts in real time. Furthermore, we examined the sensitivity of these indexes to regional ischemia. Methods and Results—The left ventricular free wall of spontaneously beating rat hearts (heart rate, 290 to 404 bpm) was directly exposed to brief high-flux, low-emittance x-ray beams provided at SPring-8. Myosin mass transfer to actin filaments was determined as the decrease in reflection intensity ratio (intensity of 1,0 plane over the 1,1 plane) between end-diastole and end-systole. The distance between 1,0 reflections was converted to a lattice spacing between myosin filaments. We found that mass transfer (mean, 1.71±0.09 SEM, n=13 hearts) preceded significant increases in lattice spacing (2 to 5 nm) during systole in nonischemic pericardium. Left coronary occlusion eliminated increases in lattice spacing and severely reduced mass transfer (P <0.01) in the ischemic region. Conclusions—Our results suggest that x-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at molecular and fiber levels that might also facilitate investigations of ventricular output regulation by the Frank-Starling mechanism.

Ghrelin Promotes Functional Angiogenesis in a Mouse Model of Critical Limb Ischemia Through Activation of Proangiogenic MicroRNAs.

Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelins proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Molecular analysis revealed that ghrelins angiogenic properties were linked to activation of prosurvival Akt/vascular endothelial growth factor/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Further, ghrelin treatment activated proangiogenic (miR-126 and miR-132) and antifibrotic (miR-30a) microRNAs (miRs) while inhibiting antiangiogenic (miR-92a and miR-206) miRs. Importantly, in vitro knockdown of key proangiogenic miRs (miR-126 and miR-132) inhibited the angiogenic potential of ghrelin. These results therefore suggest that clinical use of ghrelin for the early treatment of CLI may be a promising and potent inducer of reparative vascularization through modulation of key molecular factors.

β2-adrenergic receptor-dependent attenuation of hypoxic pulmonary vasoconstriction prevents progression of pulmonary arterial hypertension in intermittent hypoxic rats

In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4–21% O2 for 8 h/d for 6w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic β1, 2-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic β1-blocker) had no effect on the HPV in IH. These β-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the β1-adrenergic receptor (AR) was down-regulated while that of β2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of β2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the β2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.