Yumiko Hosoya

Adipogenesis in Obesity Requires Close Interplay Between Differentiating Adipocytes, Stromal Cells, and Blood Vessels

OBJECTIVE—The expansion of adipose tissue mass seen in obesity involves both hyperplasia and hypertrophy of adipocytes. However, little is known about how adipocytes, adipocyte precursors, blood vessels, and stromal cells interact with one another to achieve adipogenesis. RESEARCH DESIGN AND METHODS—We have developed a confocal microscopy-based method of three-dimensional visualization of intact living adipose tissue that enabled us to simultaneously evaluate angiogenesis and adipogenesis in db/db mice. RESULTS—We found that adipocyte differentiation takes place within cell clusters (which we designated adipogenic/angiogenic cell clusters) that contain multiple cell types, including endothelial cells and stromal cells that express CD34 and CD68 and bind lectin. There were close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. CD34+ CD68+ lectin-binding cells could clearly be distinguished from CD34− CD68+ macrophages, which were scattered in the stroma and did not bind lectin. Adipogenic/angiogenic cell clusters can morphologically and immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti–vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process. CONCLUSIONS—Living tissue imaging techniques provide novel evidence of the dynamic interactions between differentiating adipocytes, stromal cells, and angiogenesis in living obese adipose tissue.

In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

Idiopathic retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pericarditis—-Retrospective analysis of 11 case histories

Retroperitoneal fibrosis, inflammatory aortic aneurysm, and pericardial and mediastinal fibrosis are characterized by infiltration of immuno-inflammatory cells and deposition of thickened fibrous tissues. Several recent studies suggested that an immunoglobulin-G4 (IgG4)-related immunological mechanism may play a role in these diseases. By searching the clinical database of patients admitted to our department between 2000 and 2010, we summarized the clinical data of 11 patients who were diagnosed to have these disorders. The diagnoses were idiopathic retroperitoneal fibrosis (8 cases), mediastinal and/or pericardial fibrosis (4 cases), inflammatory abdominal aneurysm (2 cases), and inflammatory coronary periarteritis (1 case). Hypertension, diabetes, and dyslipidemia were found in 45%, 36%, and 55%, respectively, in these patients, and they were all either current or former smokers. Two patients with pericardial involvement showed a rushed clinical course, resulting in in-hospital death. Serum levels of IgG were elevated in 67%, and soluble interleukin-2 receptor was elevated in 75%, when measured. Immunohistochemical analysis showed marked infiltration of IgG4-positive plasma cells in the pericardium in patients who died of constrictive pericarditis. Our data support the notion that immune-inflammatory mechanism, which might be IgG4-related sometimes, may play a role in idiopathic retroperitoneal fibrosis, inflammatory aortic aneurysm, and mediastinal/pericardial fibrosis, although clinical course may differ substantially.

Responses of single-ventricular myocytes to dynamic axial stretching.

Mechano-electrical feedback (MEF) has mainly been studied in isolated single cardiomyocytes using the microelectrode and micropipette techniques, but information regarding its dynamic aspects at the cellular level is limited due to the technical difficulties associated with manipulating single cells and maintaining stable attachment of these devices. To overcome such difficulties, we have combined two experimental methods, namely a carbon fiber technique to hold single myocytes and a ratiometric fluorescence measurement technique to monitor Ca2+ transients or membrane potentials. Following an overview of the experimental technique for stretching myocytes, the results for single rat ventricular myocytes under axial stretching are presented. Ca2+ transients were influenced by the loading conditions and involvement of myofilaments was suspected in regulatory mechanism. Membrane potential measurements during dynamic axial stretching revealed that the action potential duration was prolonged when the stretch was applied during the late phase of twitch contraction, and that depolarization of the resting membrane potential depended on the phase, amplitude and speed of the applied stretch. The amplitude may also modulate the ion selectivity of stretch-activated channels. This combination of the carbon fiber technique with fluorescence measurement could represent a powerful tool for clarifying MEF at the cellular level.

Carbon fiber technique for the investigation of single-cell mechanics in intact cardiac myocytes

This protocol describes a method for attaching single isolated cardiac myocytes to carbon fibers for mechanical manipulation and measurement. This method relies on cell-adhesive carbon fibers that attach easily to the cell membrane without causing damage, and is thus applicable to intact myocytes. To connect the carbon fiber to micromanipulators, a fiber holder with glass capillaries must first be fabricated. After connection of the fibers to the micromanipulators, firm attachment is easily established by gently pressing the fiber tip onto the cell membrane. Unlike other methods, this technique does not require vast technical expertise, and therefore greatly facilitates experiments. This method enables detection of the effect of drugs, genetic defects or the expression of exogenous proteins on both active and passive properties of cardiac myocytes. In combination with other experimental procedures, this technique can also be applied to the study of mechano-transduction. This protocol can be completed in 3.5 h.

VEGF‐A induces its negative regulator, soluble form of VEGFR‐1, by modulating its alternative splicing

Vascular endothelial growth factor‐A (VEGF‐A) is one of the major angiogenic factors, and its actions are primarily mediated through its two membrane receptors, VEGFR‐1 and VEGFR‐2. A soluble form of VEGFR‐1 (sVEGFR‐1) sequesters the free form of VEGF‐A, and acts as a potent anti‐angiogenic factor. While sVEGFR‐1 is synthesized as a splice variant of VEGF‐R1 gene, the interactions between VEGF‐A and sVEGFR‐1 remain largely unknown. Here, we show that VEGF‐A upregulates sVEGF‐R1 expression in human vascular endothelial cells but leaves full‐length VEGF‐R1 expression unchanged, and that this induction was dependent on the VEGFR‐2‐protein kinase C‐MEK signaling pathway. The VEGF‐A‐induced sVEGFR‐1 upregulation can operate as a negative feedback system, which if modulated can become a novel therapeutic target for regulating pathological angiogenesis.

Angiotensin II impairs endothelial nitric-oxide synthase bioavailability under free cholesterol-enriched conditions via intracellular free cholesterol-rich membrane microdomains.

Background: Free cholesterol forms membrane microdomains in endothelial cells. Results: Free cholesterol loading formed intracellular vesicles with free cholesterol-rich microdomains that were shifted toward late endosomes/lysosomes by angiotensin II, resulting in impairment of endothelial NOS availability. Conclusion: Angiotensin II decreased the bioavailability of endothelial NOS in a free cholesterol-enriched condition. Significance: Free cholesterol-rich microdomains are crucial platforms of endothelial NOS availability. Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.

A Case of Localized IgG4-Related Thoracic Periarteritis and Recurrent Nerve Palsy

Periarteritis, including periaortitis, is a systemic disorder characterized by an excessive fibroinflammatory reaction that can result in the compromise of great vessels and periarterial/periaortic structures. Recent studies have suggested that IgG4-related inflammation may play a role in chronic periaortitis. These pathologic conditions might represent a systemic disorder with fibrotic reaction rather than local inflammation. In this report, the authors describe a case of a 31-year-old man with marked periaortic fibrous thickening localized to the aortic arch, which was histologically and serologically proven to be IgG4 related. Positron emission tomography showed increased ¹⁸F-fluorodeoxyglucose uptake at this region. Histologic examination revealed infiltration of lymphoplasmacytes and marked fibrosis with numerous IgG4-positive plasma cells. The serum concentration of IgG4 was 263 mg/dL. The size of the periaortic mass and ¹⁸F-fluorodeoxyglucose uptake at this region markedly decreased under corticosteroid therapy. This case suggests that IgG4-related periarteritis can also occur as a solitary focus in the cardiovascular system.

Plasma neutrophil gelatinase-associated lipocalin predicts major adverse cardiovascular events after cardiac care unit discharge

BACKGROUND Emerging acute kidney injury biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), have a high potential for predicting worsening renal function. Acute exacerbation of renal dysfunction has a great impact on the outcomes of cardiovascular patients in critical conditions. This study aimed to evaluate whether plasma NGAL can predict the mortality and major adverse cardiovascular events (MACEs) after discharge from the cardiac care unit (CCU). METHODS Patients who were admitted to the CCU of the Tokyo University Hospital were prospectively enrolled (101 patients). Blood and urinary markers, including the blood NGAL, brain natriuretic peptide, creatinine, cystatin C, urinary albumin, N-acetyl-β-d-glucosaminidase, and L-type fatty acid-binding protein, were measured at CCU discharge. The primary outcome was MACEs until at least 6 months after CCU discharge. RESULTS Thirty-five patients experienced MACEs (35%). Multivariate logistic analysis revealed that the plasma NGAL, length of CCU stay, and existence of diabetes and heart failure were independent predicting factors for MACEs. Patients with the highest NGAL at discharge (>75th percentile) showed a significantly higher risk of MACEs than those with the lowest NGAL (<25th percentile) (log-rank test; hazard ratio, 5.15; 95% confidence interval 1.84-18.20; p<0.01). CONCLUSION Plasma NGAL at CCU discharge is a significant prognostic indicator of outcomes at 6 months in critically ill cardiac patients treated in a CCU.

Effect of add-on aliskiren to type 1 angiotensin receptor blocker therapy on endothelial function and autonomic nervous system in hypertensive patients with ischemic heart disease.

The aim of this study was to evaluate the add‐on effect of aliskiren to valsartan on endothelial‐dependent vasodilation in hypertensive patients with ischemic heart disease (IHD). After 4 weeks of treatment with 80 mg of valsartan, 28 patients were allocated to either continued treatment with valsartan or an add‐on treatment with valsartan plus 150 mg of aliskiren. Aliskiren significantly decreased plasma renin activity, whereas endothelium‐dependent vasodilation measured by flow‐mediated dilation (FMD) did not change. In contrast, heart rate significantly decreased (73.1 ± 9.8 to 66.3 ± 7.0 beats per minute at baseline and 24 weeks, respectively [P = .009]) and the standard deviation of the R‐R intervals (SDNN) significantly increased in the aliskiren group. The add‐on aliskiren to valsartan therapy may not improve endothelial functions, although it significantly reduced resting heart rate via regulation of the autonomic nervous system in hypertensive patients with IHD.