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Statins and therapeutic ultrasound (TUS) have been shown to ameliorate angiogenesis on ischemic hindlimb animals and promote human umbilical vein endothelial cells (HUVECs) tube formation and proliferation. Here, we evaluate the therapeutic effect of TUS in combination with atorvastatin (Ator) therapy on angiogenesis in hindlimb ischemia and HUVECs. After subjecting excision of the left femoral artery, all mice were randomly distributed to one of four groups: Control; Ator treated mice (Ator); TUS treated mice (TUS); and Ator plus TUS treated mice (Ator+TUS). At day 14 post-surgery, the Ator plus TUS treatment cohort had the greatest blood perfusion, accompanied by elevated capillary density. In vitro, Ator plus TUS augmented tube formation, migration and proliferative capacities of HUVECs. Additionally, the united administration upregulated expression of angiogenic factors phosphorylated Akt (p-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor (VEGF), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. Our data indicated that the united administration could significantly enhance ischemia-mediated angiogenesis and exert a protective effect against ischemic/hypoxia induced damage among HUVECs through up-regulating VEGF expression and activating the PI3K-Akt-eNOS pathway., Competing Interests: None.

[This corrects the article on p. 2068 in vol. 10, PMID: 30093944.].

Nanostructured calcium phosphate (CaP) and magnesium phosphate (MgP) are promising for the application as the nanocarriers in drug delivery. However, the difference between CaP and MgP nanocarriers in drug delivery is rarely investigated. In this work, we comparatively investigated nanostructured CaP, MgP and calcium magnesium phosphate (CMP) for the delivery of SRT1720, which is a silent information regulator (SIRT1) specific activator with pro-angiogenic and anti-aging properties in response to hydrogen peroxide (H 2 O 2 )-induced endothelial senescence. The protection of SRT1720-loaded CaP nanospheres, MgP nanosheets and CMP microspheres on the H 2 O 2 -induced senescent endothelium was examined by using human umbilical vein endothelial cells (HUVECs), demonstrating the improved cell viability, anti-aging, tube formation and migration. In addition, the SRT1720-loaded CaP nanospheres, MgP nanosheets and CMP microspheres can rescue the impaired angiogenic potential of HUVECs via activation of Akt/eNOS/VEGF pathway. The SRT1720-loaded MgP nanosheets and CMP microspheres have a similar protective effect compared with the pure SRT1720, while the SRT1720-loaded CaP nanospheres decrease the protective capability of SRT1720. These results lead us to figure out both MgP nanosheets and CMP microspheres are suitable and effective delivery for SRT1720 and this system can be further applied in vivo treatment., Competing Interests: None.

The objective of this investigation was to evaluate the therapy effect of combined therapeutic ultrasound (TUS) treatment and pulsed electromagnetic field (PEMF) therapy on angiogenesis in hypertension-related hindlimb ischemia. After subjecting excision of the left femoral artery, spontaneously hypertensive rats (SHRs) were randomly distributed to one of four groups: SHR; TUS treated SHR (SHR-TUS); PEMF treated SHR (PEMF-TUS); and TUS plus PEMF treated SHR (SHR-TUS-PEMF). Wistar-Kyoto rats (WKYs) with femoral artery excision were regarded as a control group. At day 14 after surgery, the TUS plus PEMF united administration had the greatest blood perfusion accompanied by elevated capillary density and the lowest TUNEL index. Interestingly, the united administration up-regulated the angiogenic factors expression of phosphorylated Akt (p-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS), vascular endothelial growth factor (VEGF), anti-apoptotic protein of Bcl-2 and down-regulated pro-apoptotic protein levels of Bax and Cleaved caspase-3 in vivo . Our results demonstrated that the united administration could significantly rescue hypertension-related inhibition of ischemia-induced neovascularization partly by promoting angiogenesis and inhibiting apoptosis., Competing Interests: None.

Background: Previous studies have demonstrated that therapeutic ultrasound (TUS) ameliorates angiogenesis on ischemic hind limb animals and also promotes human umbilical vein endothelial cells (HUVECs) tube formation. Apoptosis plays a key role in post-ischemic angiogenesis pathogenesis. However, the mechanisms underlying the anti-apoptotic effects of TUS are not clear. Therefore we put forward the hypothesis that TUS might promote angiogenesis during ischemia/hypoxia (I/H) by decreasing apoptosis. Methods: We investigated the cytoprotective role of TUS and the underlying mechanisms in I/H-induced HUVEC apoptosis. HUVECs were treated under hypoxic serum-starved conditions for 36 h and then treated with or without TUS (9 minutes, 1 MHz, 0.3 W/cm 2 ). The cell viability was examined by the CCK-8 assay, apoptosis cell rate was determined by TUNEL staining and flow cytometry assay. In addition, the mitochondrial-dependent apoptosis pathway was evaluated by the protein activity of Bax, Bcl-2 and Caspase-3. Results: 1) apoptosis could be induced by I/H in HUVECs. 2) TUS attenuates HUVECs cell apoptosis induced by I/H. 3) TUS inhibits the protein expression of apoptosis modulators and effectors that regulate the mitochondrial pathway of apoptosis in HUVECs. 4) TUS increases the phosphorylation of Akt, which demonstrates the activation of the phosphoinositide 3-kinase (PI3K)- serine/threonine kinase (Akt) signal pathway. Conclusions: The present study indicates that exposure to TUS exerts a protective effect against I/H-induced apoptosis among HUVECs and that this process is mediated through the mitochondrial-dependent intrinsic apoptotic pathway. We also confirm that the PI3K-Akt signal cascade may be taken part in the TUS effects on apoptosis., Competing Interests: None.

Therapeutic ultrasound (TUS) has been demonstrated to improve endothelial nitric oxide synthase (eNOS) activity, which played a crucial role in the regulation of angiogenesis. Diabetes Mellitus (DM) impairs eNOS activity. We tested the hypothesis that DM may retard unilateral hindlimb ischemia-induced angiogenesis by inhibiting eNOS in high-fat diet (HFD)/streptozocin (STZ) induced diabetic mice, and that TUS may reverse DM-related impairment of angiogenesis. C57BL/6 mice were allocated to four groups: (A) mice were fed standard diet (control); (B) mice were fed standard diet and treated with TUS (control+TUS); (C) type-2 DM mice were induced by HFD/STZ (diabetic); and (D) type-2 DM mice and treated with TUS (dabetic+TUS). All mice were surgically induced unilateral limb ischemia. The ischemic skeletal muscles in groups B and D were irradiated with extracorporeal TUS for 9 minutes/day (frequency of 1 MHz, intensity of 0.3 W/cm 2 ) for 14 consecutive days. The result showed that TUS augmented the blood perfusion, increased capillary density accompanied by an upregulation of angiogenic factors and a downregulation of apoptotic proteins in group D relative to group C. In vitro , TUS inhibited the apoptosis, promoted tubule formation, proliferation and migration capacities, increased angiogenic factors expression and reduced apoptotic protein levels in human umbilical vein endothelial cells (HUVECs). Furthermore, TUS can robust reverse the inhibiting effect induced by high glucose (HG) on HUVECs, and these benefits could be blocked by phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) or eNOS inhibitor (L-NAME). Together, TUS restored type-2 DM-mediated inhibition of ischemia-induced angiogenesis, partially via PI3K-Akt-eNOS signal pathway.

Silent information regulator 1 (SIRT1) plays a critical role in maintaining vascular homeostasis via modulating senescent-related signal pathway, however, the molecular mechanism remains modest clarified. The purpose of this study was to examine whether SIRT1 specific activator SRT1720 would exhibit pro-angiogenic and anti-aging properties in response to hydrogen peroxide (H2O2)-induced endothelial senescence, and determine the underlying mechanisms. We pre-treated senescent human umbilical vein endothelial cells (HUVECs) with SRT1720, senescence-associated beta-galactosidase activity, apoptosis, migration, tube formation, proliferation and angiogenic factors were quantitatively examined. The results revealed that pharmacologic activation of SIRT1 by SRT1720 rescued apoptotic HUVECs and upregulated angiogenic response through reinforcing the protein expressions of angiogenic and survival factors in vitro. Furthermore, we confirmed that the expressions of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and phosphoryl-Akt were augmented in SRT1720-treated senescent HUVECs. In conclusion, our data indicated that SRT1720 could protect against endothelial senescence and maintain cell function via Akt/eNOS/VEGF axis.

We examined the hypothesis that therapeutic ultrasound (TUS) treatment would rescue the hypertension-related inhibition of ischemia-induced angiogenesis. TUS protects against endothelial dysfunction, but it is little known that the effect of TUS treatment on angiogenesis inhibited by hypertension. 20-week-old male spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) were randomly allocated to 4 groups: SHR; TUS treated SHR (SHR-TUS); WKY and TUS treated WKY (WKY-TUS). After undergoing excision of the left femoral artery, the ischemic skeletal muscles were treated with extracorporeal TUS for 9 minutes of daily exposure (frequency of 1 MHz, intensity of 0.3 W/cm(2)) for 14 consecutive days. We found that TUS normalized the blood perfusion in SHR-TUS accompanied by elevated capillary density. Similar results were found in the protein expression of angiogenic factors. TUS treatment also enhanced peripheral capillary density in WKY rats and restored the capillary rarefaction in hypertension by elevating the protein levels of endothelial nitric oxide synthase (eNOS), hypoxic inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and phosphorylated Akt (p-Akt) in vivo. Our data demonstrated that TUS treatment ameliorated hypertension-related inhibition of ischemia-induced angiogenesis, at least in part, via an NO-dependent manner.

Therapeutic angiogenic effects of low-intensity ultrasound have been reported in endothelial cells and animal models of hind limb ischemia. It has been shown that the proliferation, migration, and tube formation of endothelial cells play critical roles in angiogenesis. The purpose of this study was to determine the underlying mechanism of low-intensity continuous therapeutic ultrasound on angiogenesis in endothelial cells. In the present study, human umbilical vein endothelial cells (HUVECs) were simulated of low-intensity therapeutic ultrasound (TUS, 1 MHz, 0.3 W/cm(2), 9 minute per day) for 3 days, and we observed migration, tube formation, and expression of endothelial nitric oxide synthase (eNOS) and serine/threonine kinase (Akt) in HUVECs. Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and TUS-induced changes in the pathways that mediate angiogenesis were investigated. After exposure to TUS, HUVECs tube formation and migration were significantly promoted, which was blocked by the eNOS inhibitor Immunofluorescence assay and Western blotting analysis demonstrated that eNOS expression in the HUVECs was significantly increased after TUS exhibition. Proteins of phosphorylated eNOS and Akt were both up-regulated after TUS stimulation. However, the specific inhibitor of PI3K not only significantly decreased the expression of p-Akt, but also down-regulated the p-eNOS. This suggested that the PI3K/Akt signal pathway might participate in modulating the activity of eNOS. In short, TUS therapy promotes angiogenesis through activation of the PI3K-Akt-eNOS signal cascade in HUVECs.

The diagnosis of uterine smooth muscle tumors including leiomyosarcomas (LMS), smooth muscle tumors of uncertain malignant potential (STUMP), bizarre (atypical) leiomyoma (BLM), mitotically active leiomyoma (MAL) and leiomyoma (LM) depends on a combination of microscopic features, such as mitoses, cytologic atypia, and coagulative tumor cell necrosis. However, a small number of these tumors still pose difficult diagnostic challenges. The assessment of accurate mitotic figures (MF) is one of the major parameters in the proper classification of uterine smooth muscle tumors. This assessment can be hampered by the presence of increased number of apoptotic bodies or pyknotic nuclei, which frequently mimic mitoses. Phospho-histone H3 (PHH3) is a recently described immunomarker specific for cells undergoing mitoses. In our study, we collected 132 cases of uterine smooth muscle tumors, including 26 LMSs, 16 STUMPs, 30 BLMs, 30 MALs and 30 LMs. We used mitosis specific marker PHH3 to count mitotic indexes (MI) of uterine smooth muscle tumors and compared with the mitotic indexes of hematoxylin and eosin (H&E). There is a positive correlation with the number of mitotic figures in H&E-stained sections and PHH3-stained sections (r=0.944, P<0.05). The ratio of PHH3-MI to H&E-MI has no statistically significant difference in each group except for LMs (P>0.05). The counting value of PHH3 in LMSs have significantly higher than STUMPs, BLMs, MALs and LMs (P<0.001) and the counting value of PHH3 is 1.5±0.5 times of the number of mitotic indexes in H&E. To conclude, our results show that counting PHH3 is a useful index in the diagnosis of uterine smooth muscle tumors and it can provide a more accurate index instead of the time-honored mitotic figure counts at a certain ratio.

Pulsed electromagnetic fields (PEMF) have been shown to promote proliferation and regeneration in the damaged tissue. Here, we examined whether PEMF therapy improved postnatal neovascularization using murine model of hindlimb ischemia, and the underlying cellular/molecular mechanisms were further investigated. Hindlimb ischemia was induced by unilateral femoral artery resection using 6-8 week-old male C57BL6 mice. Then, mice were exposed to extracorporeal PEMF therapy (4 cycles, 8min/cycle, 30 ± 3 Hz, 5 mT) every day until day 14. Our data demonstrated that PEMF therapy significantly accelerated wound healing, decreased prevalence of gangrene and increased postnatal neovascularization. Moreover, the levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and Akt phosphorylation in ischemic muscles were markedly enhanced following PEMF therapy. In vitro, PEMF inhibited the process of hypoxia-induced apoptosis and augmented tube formation, migration and proliferative capacities of human umbilical vein endothelial cells (HUVECs). Additionally, PEMF exposure increased VEGF secretion, as well as the eNOS and Akt phosphorylation, and these benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. In conclusion, our data indicated that PEMF therapy enhanced ischemia-mediated angiogenesis, through up-regulating VEGF expression and activating the PI3K-Akt-eNOS pathway. Therefore, PEMF should be a valuable treatment for the patients with critical limb ischemia.

Malignant germ cell tumors of the ovary are very rare and account for about 2-5% of all ovarian tumors of germ origin. Most patients are adolescent and young women, approximately two-thirds of them are under 20 years of age, occasionally in postmenopausal women. But clear cell carcinoma usually occurs in older patients (median age: 57-year old), and closely related with endometriosis. Here we report a case of a 55-year old woman with right ovarian mass that discovered by B ultrasonic. Her serum levels of human chorionic gonadotropin (hCG) and α-fetoprotein (AFP) were elevated. Pathological examination revealed the tumor to be a mixed germ cell tumor (yolk sac tumor, embryonal carcinoma and mature teratoma) with clear cell carcinoma in a background of endometriosis. Immunohistochemical staining showed SALL4 and PLAP were positive in germ cell tumor area, hCG, CD30 and OCT4 were positive in epithelial-like cells and giant synctiotrophoblastic cells, AFP, AAT, CD117 and Glyp3 were positive in yolk sac component, EMA and CK7 were positive in clear cell carcinoma, CD10 was positive in endometrial cells of endometriotic area. She was treated with surgery followed by seven courses of chemotherapy. She is well and serum levels of hCG and AFP have been decreased to normal levels.

Although significant progress in bypass surgery and catheter intervention against peripheral artery disease, the number of severe critical limb ischemia (CLI) patients is increasing. Thus, it is crucial to develop new, non-invasive therapeutic strategies. The purpose of this study was to determine the mechanism of therapeutic ultrasound (TUS) on ischemic angiogenesis using mouse model of hindlimb ischemia and the cellular/molecular mechanisms underlying TUS-related neovascularization. The hindlimb ischemic mice were exposed to extracorporeal TUS for 3, 6, 9 minute per day (1 MHz, 0.3 W/cm(2)) until day 14 after left femoral artery ligation. Increased blood perfusion and capillary density were determined following 9 min of TUS compared with ischemic group. Moreover, TUS treatment increased the protein levels of vascular endothelial growth factor (VEGF), hypoxic inducible factor-1α (HIF-1α), endothelial nitric oxide synthase (eNOS) and p-Akt in vivo. TUS promoted capillary-like tube formation, migration and motility of human umbilical venous endothelial cells (HUVECs). Furthermore, the protein expressions of VEGF, eNOS and p-Akt were increased after TUS treatment. In conclusion, TUS therapy promotes postnatal neovascularization through multiple angiogenic pathways in mice model of ischemic hindlimb.

Extracorporeal pulsed electromagnetic field (PEMF) has been shown the ability to improve regeneration in various ischemic episodes. Here, we examined whether PEMF therapy facilitate cardiac recovery in rat myocardial infarction (MI), and the cellular/molecular mechanisms underlying PEMF-related therapy was further investigated. The MI rats were exposed to active PEMF for 4 cycles per day (8 minutes/cycle, 30 ± 3 Hz, 5 mT) after MI induction. The data demonstrated that PEMF treatment significantly inhibited cardiac apoptosis and improved cardiac systolic function. Moreover, PEMF treatment increased capillary density, the levels of vascular endothelial growth factor (VEGF) and hypoxic inducible factor-1α in infarct border zone. Furthermore, the number and function of circulating endothelial progenitor cells were advanced in PEMF treating rats. In vitro, PEMF induced the degree of human umbilical venous endothelial cells tubulization and increased soluble pro-angiogenic factor secretion (VEGF and nitric oxide). In conclusion, PEMF therapy preserves cardiac systolic function, inhibits apoptosis and trigger postnatal neovascularization in ischemic myocardium.

Hypertension is considered as one of the major risk factors of atherosclerosis, especially for carotid artery plaque, which is a sign for cardiovascular incapacity and cerebral infarction. As adult age, systolic blood pressure (SBP or S) tends to rise and diastolic blood pressure (DBP or D) tends to fall, thus the pulse pressure (PP) will increase. The vascular injury was directly proportional to the level of SBP, and inversely proportional to DBP. But so far, studies of the vascular injury based on SBP and DBP measurement were mostly qualitative. The exact contribution of each parameter to the vascular injury has not been quantitatively identified. In this study, we employed a mathematical model to predict the risk for plaques of carotid arteries in aged people and combined the SBP, DBP and heart rate (HR) to perform a quantitative analysis. We analyzed 1672 males who were over 60-year-old and hospitalized due to atherosclerosis-related diseases and received a 24-h arterial blood pressure monitoring (ABPM) examination. These patients were divided into 19 subgroups using the ABPM data, 24-h average SBP, DBP and HR as variables based on the ascending order of the magnitude of each element. We developed a new index, namely the dynamic level (DL) which correlated best with the plaque formation of carotid arteries among all the well-established indexes for blood pressure. We demonstrated that index DL has better correlation to plaques incidence tendency (p < 0.0001) when compared to either SBP (P < 0.05) or PP (P < 0.001) alone. The risk on incidence of the plaques of carotid arteries has positive correlation with first power of SBP and -0.8 power of DBP. This model can be used clinically to predict the occurrence of plaque formation.