Haruki Sekiguchi

CXCR4 blockade augments bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC–mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.

The Relative Potency and Safety of Endothelial Progenitor Cells and Unselected Mononuclear Cells for Recovery From Myocardial Infarction and Ischemia

Endothelial progenitor cells (EPCs) are a subset of the total mononuclear cell population (tMNCs) that possess an enhanced potential for differentiation within the endothelial‐cell lineage. Typically, EPCs are selected from tMNCs via the expression of both hematopoietic stem‐cell markers and endothelial‐cell markers, such as CD34, or by culturing tMNCs in media selective for endothelial cells. Both EPCs and tMNCs participate in vascular growth and regeneration, and their potential use for treatment of myocardial injury or disease has been evaluated in early‐phase clinical studies. Direct comparisons between EPCs and tMNCs are rare, but the available evidence appears to favor EPCs, particularly CD34+ cells, and the potency of EPCs may be increased as much as 30‐fold through genetic modification. However, these observations must be interpreted with caution because clinical investigations of EPC therapy are ongoing. We anticipate that with continued development, EPC therapy will become a safe and effective treatment option for patients with acute myocardial infarction or chronic ischemic disease. J. Cell. Physiol. 219: 235–242, 2009.

CXCR4 Antagonist AMD3100 Accelerates Impaired Wound Healing in Diabetic Mice

The antagonism of CXC-chemokine receptor 4 (CXCR4) with AMD3100 improves cardiac performance after myocardial infarction by augmenting the recruitment of endothelial progenitor cells (EPCs) from the bone marrow to the regenerating vasculature. We investigated whether AMD3100 may accelerate diabetes-impaired wound healing through a similar mechanism. Skin wounds were made on the backs of leptin-receptor–deficient mice and treated with AMD3100 or saline. Fourteen days after treatment, wound closure was significantly more complete in AMD3100-treated mice (AMD3100: 87.0±2.6%, Saline: 33.1±1.8%; P<0.0001) and was accompanied by greater collagen-fiber formation, capillary density, smooth-muscle-containing vessel density, and monocyte/macrophage infiltration. On day 7 after treatment, AMD3100 was associated with higher circulating EPC and macrophage counts and with significantly upregulated mRNA levels of stromal-cell–derived factor 1 and platelet-derived growth-factor B in the wound bed. AMD3100 also promoted macrophage proliferation and phagocytosis and the migration and proliferation of diabetic mouse primary dermal fibroblasts and 3T3 fibroblasts, which express very little CXCR4. In conclusion, a single topical application of AMD3100 promoted wound healing in diabetic mice by increasing cytokine production, mobilizing bone-marrow EPCs, and enhancing the activity of fibroblasts and monocytes/macrophages, thereby increasing both angiogenesis and vasculogenesis. Not all of the AMD3100-mediated effects evolved through CXCR4 antagonism.

CXC-Chemokine Receptor 4 Antagonist AMD3100 Promotes Cardiac Functional Recovery After Ischemia/Reperfusion Injury via Endothelial Nitric Oxide Synthase–Dependent Mechanism

Background— CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia/reperfusion injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed. Methods and Results— Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after ischemia/reperfusion injury. Three days later, histological measurements of the ratio of infarct area to area at risk were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4+ cells were mobilized for just 1 day in both groups, but the mobilization of sca1+/flk1+ cells endured for 7 days in AMD3100-treated mice compared with just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and 2 targets of eNOS signaling, matrix metalloproteinase-9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1+/flk1+ cell mobilization without altering the mobilization of CXCR4+ cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice but not in wild-type mice with eNOS-knockout BM. Conclusions— AMD3100 prolongs BM progenitor mobilization and improves recovery from ischemia/reperfusion injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.

Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse.

Randomized pilot trial comparing tolvaptan with furosemide on renal and neurohumoral effects in acute heart failure

Loop diuretics are first‐line medications for congestive heart failure (CHF); however, they are associated with serious adverse effects, including decreased renal function, and sympathetic nervous and renin–angiotensin system activation. We tested whether tolvaptan, a vasopressin V2‐receptor antagonist, could reduce unfavourable furosemide‐induced effects during CHF treatment.

Estradiol promotes neural stem cell differentiation into endothelial lineage and angiogenesis in injured peripheral nerve

Neural stem cells (NSCs) differentiate into endothelial cells (ECs) and neuronal cells. Estradiol (E2) is known to exhibit proangiogenic effects on ischemic tissues via EC activation. Therefore, we hypothesized that E2 can promote the therapeutic potential of NSC transplantation for injured nerve repair via the differentiation of NSCs into ECs during neovascularization. NSCs isolated from newborn mouse brains were transplanted into injured sciatic nerves with (NSC/E2 group) or without E2-conjugated gelatin hydrogel (E2 group). The NSC/E2 group exhibited the greatest recovery in motor nerve conduction velocity, voltage amplitude, and exercise tolerance. Histological analyses revealed increased intraneural vascularity and blood perfusion as well as striking NSC recruitment to the neovasculature in the injured nerves in the NSC/E2 group. In vitro, E2 enhanced the NSC migration and proliferation inhibiting apoptosis. Fluorescence-activated cell sorting analysis also revealed that E2 significantly increased the percentage of CD31 in NSCs, and the effect of E2 was completely neutralized by the estrogen receptor antagonist ICI. The combination of E2 administration and NSC transplantation cooperatively improved the functional recovery of injured peripheral nerves, at least in part, via E2-associated NSC differentiation into ECs. These findings provide a novel mechanistic insight into both NSC biology and the biological effects of endogenous E2.

Estradiol triggers sonic-hedgehog-induced angiogenesis during peripheral nerve regeneration by downregulating hedgehog-interacting protein

Both estradiol (E2) and Sonic Hedgehog (Shh) contribute to angiogenesis and nerve regeneration. Here, we investigated whether E2 improves the recovery of injured nerves by downregulating the Shh inhibitor hedgehog-interacting protein (HIP) and increasing Shh-induced angiogenesis. Mice were treated with local injections of E2 or placebo one week before nerve-crush injury; 28 days after injury, nerve conduction velocity, exercise duration, and vascularity were significantly greater in E2-treated mice than in placebo-treated mice. E2 treatment was also associated with higher mRNA levels of Shh, the Shh receptor Patched-1, and the Shh transcriptional target Gli1, but with lower levels of HIP. The E2-induced enhancement of nerve vascularity was abolished by the Shh inhibitor cyclopamine, and the effect of E2 treatment on Shh, Gli1, and HIP mRNA expression was abolished by the E2 inhibitor ICI. Gli-luciferase activity in human umbilical-vein endothelial cells (HUVECs) increased more after treatment with E2 and Shh than after treatment with E2 alone, and E2 treatment reduced HIP expression in HUVECs and Schwann cells without altering Shh expression. Collectively, these findings suggest that E2 improves nerve recovery, at least in part, by reducing HIP expression, which subsequently leads to an increase in Shh signaling and Shh-induced angiogenesis.

Estimation of parasympathetic nerve function during sleep in patients with obstructive sleep apnea by instantaneous time–frequency analysis

BACKGROUND AND OBJECTIVES The pathophysiologic aspects of parasympathetic nerve (PN) function during sleep in patients with obstructive sleep apnea (OSA) studied by classical power spectrum analysis on heart rate variability (HRV) are highly controversial. The controversy is attributed to methodologic concerns, such as poor time resolution involved in power spectrum analysis. We aimed to establish the appropriate method for the investigation of PN function in OSA patients with apneas and hypopneas using instantaneous time-frequency analysis with complex demodulation (CD) and sufficient time resolution. METHODS A total of 30 patients with PSG-confirmed mild to severe OSA were recruited for the analysis of frequency spectra contained in R-R intervals (RRI) of overnight electrocardiograph (ECG) tracings. High-frequency (HF) domains ranging between 0.15 and 0.40 Hz were selected for analysis. Among these domains, the HF domain with the maximum instantaneous amplitude was defined as the main HF peak and was used as the surrogate marker of PN discharge. Based on density spectrum array (DSA) map for main HF peak constructed with a time scale of 1s and a frequency resolution of 0.002 Hz (HF-DSA map), the shift in central frequency (CF) of main HF peak over time was continuously monitored. When the main HF peak with the same CF lasted for more than 20 s or 5 min on HF-DSA map, the PN function was considered to be stable or very stable. The measurements were then repeated after continuous positive airway pressure (CPAP) treatment. RESULTS The extent of PN-evoked modulation of RRI was enhanced in nonrapid eye movement (NREM) sleep, though the stability was reduced in both NREM and rapid eye movement (REM) sleep. These peculiar behaviors of PN function were reversed by CPAP treatment. CONCLUSION We found that instantaneous time-frequency analysis allowed estimation of transitional changes in PN function during sleep in OSA patients.

Gender-Specific Impacts of Apnea, Age, and BMI on Parasympathetic Nerve Dysfunction during Sleep in Patients with Obstructive Sleep Apnea

Background The gender-specific influences of various confounding factors, including apnea, age, BMI, and cigarette consumption, on the function of the parasympathetic nerve system (PNS) during sleep in OSA patients has never been investigated. Methods One hundred ninety-seven males and 63 females with OSA were subjected to full PSG examinations including assessment of R-R intervals (RRIs) during an overnight ECG. The PNS-derived modulatory effect on the RRIs and the variability of this effect were quantified during REM and NREM using instantaneous time-frequency analysis with complex demodulation. The spectral domain with the maximum instantaneous amplitude in the high-frequency band between 0.15 and 0.4 Hz was defined as the main HF peak and used as a surrogate marker of PNS discharge. Based on density-spectrum-array maps of the main HF peaks (HF-DSA map), shifts in the central frequency of the main HF peak over time were continuously observed. When the main HF peaks on the HF-DSA maps maintained the same central frequency for more than 20 sec or 5 min, the PNS functions were considered to be “stable” or “very stable”, respectively. Results Apneas enhanced PNS-derived cardiac-modulation during REM in males, but more importantly, they made PNS-function unstable during both REM and NREM in males and during NREM in females. Aging blunted the PNS-derived cardiac-modulation during both REM and NREM regardless of gender, but aging had no impact on the stability of PNS-function. BMI blunted PNS-eliciting cardiac-modulation during REM in males and during NREM in both males and females. BMI made the PNS unstable during REM in females. Neither height nor cigarette consumption influenced any PNS-related parameter. Conclusions The PNS-derived cardiac-modulation was generally inhibited by aging and obesity, in which the effect of obesity was gender-specific. The PNS instability at nighttime was mainly induced by apneas but by obesity particularly during REM in females.