Perinatal hypoxia aggravates occlusive pulmonary vasculopathy in SU5416/hypoxia-treated rats later in life.

Pulmonary arterial hypertension (PAH) is a fatal disease, which is characterized by occlusive pulmonary vascular disease (PVD) in small pulmonary arteries. It remains unknown whether perinatal insults aggravate occlusive PVD later in life. We tested the hypothesis that perinatal hypoxia aggravates PVD and survival in rats. PVD was induced in rats with/without perinatal hypoxia (embryonic day 14 to postnatal day 3) by injecting SU5416 at 7 wk of age and subsequent exposure to hypoxia for 3 wk (SU5416/hypoxia). Hemodynamic and morphological analyses were performed in rats with/without perinatal hypoxia at 7 wk of age (baseline rats, n = 12) and at 15 wk of age in 4 groups of rats: SU5416/hypoxia or control rats with/without perinatal hypoxia (n = 40). Pulmonary artery smooth muscle cells (PASMCs) from the baseline rats with/without perinatal hypoxia were used to assess cell proliferation, inflammation, and genomic DNA methylation profile. Although perinatal hypoxia alone did not affect survival, physiological, or pathological parameters at baseline or at the end of the experimental period in controls, perinatal hypoxia decreased weight gain and survival rate and increased right ventricular systolic pressure, right ventricular hypertrophy, and indices of PVD in SU5416/hypoxia rats. Perinatal hypoxia alone accelerated the proliferation and inflammation of cultured PASMCs from baseline rats, which was associated with DNA methylation. In conclusion, we established the first fatal animal model of PAH with worsening hemodynamics and occlusive PVD elicited by perinatal hypoxia, which was associated with hyperproliferative, proinflammatory, and epigenetic changes in cultured PASMCs. These findings provide insights into the treatment and prevention of occlusive PVD.

CRISPR-mediated Bmpr2 point mutation exacerbates late pulmonary vasculopathy and reduces survival in rats with experimental pulmonary hypertension.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (Bmpr2) mutations present earlier with severe hemodynamic compromise and have poorer survival outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with Bmpr2 mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant Bmpr2 mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats. METHODS: A monoallelic single nucleotide insertion in exon 1 of Bmpr2 (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed. RESULTS: The +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/[LV + S]) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type. CONCLUSIONS: The present study demonstrates that the Bmpr2 mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.

Lung damage created by high tidal volume ventilation in rats with monocrotaline-induced pulmonary hypertension.

BACKGROUND: Rats with chronic hypoxia-induced non-inflammatory pulmonary hypertension (PH) are resistant to ventilator-induced lung injury. We investigated the effect of high tidal volume ventilation in another model of PH, monocrotaline (MCT)-induced PH, which is a type of inflammatory PH. METHODS: PH was induced in rats by subcutaneous injection with 60 mg/kg MCT. Normal control rats, rats at 2 weeks after MCT injection (MCT2), and rats at 3 weeks after MCT injection (MCT3) were ventilated with low tidal volume (LV, 6 mL/kg) or high tidal volume (HV, 35 mL/kg) for 2 h with room air without positive end-expiratory pressure. Arterial oxygen pressure (PaO2) and Evans blue dye (EBD) extravasation were measured. Hypertensive pulmonary vascular remodeling was assessed morphometrically by the percentage of muscularized peripheral pulmonary arteries (%Muscularization) and the media wall thickness to external diameter ratio, namely percentage medial wall thickness (%MWT). To assess inflammation, lung IκB protein and cytokine mRNA expression levels were assessed. RESULTS: Baseline mean pulmonary arterial pressure was significantly higher in MCT rats (normal, 15.4 ± 0.5 mmHg; MCT2, 23.7 ± 0.9; and MCT3, 34.5 ± 1.5). After 2-h ventilation, PaO2 was significantly lower in the HV groups compared with the LV groups in normal and MCT2 rats, but not in MCT3 rats. Impairment of oxygenation with HV was less in MCT3 rats compared with normal and MCT2 rats. Among the HV groups, MCT3 rats showed significantly lower levels of EBD extravasation than normal and MCT2 rats. HV significantly downregulated IκB protein expression in normal and MCT3 rats and increased IL-6, MCP-1, CXCL-1 (MIP-1), and IL-10 mRNA levels in MCT3 rats. %Muscularization, %MWT, and the expression of lung elastin were significantly higher in MCT3 rats than in normal and MCT2 rats. CONCLUSION: We found that HV-associated damage might be reduced in MCT-induced PH rats compared with normal rats. The results of this and earlier studies suggest that hypertensive pulmonary vascular structural changes might be protective against the occurrence of ventilator-induced lung injury, irrespective of the etiology of PH.

Enhanced antitumor activity of combined lipid bubble ultrasound and anticancer drugs in gynecological cervical cancers.

Chemotherapy plays an important role in the treatment of patients with gynecological cancers. Delivering anticancer drugs effectively to tumor cells with just few side effects is key in cancer treatment. Lipid bubbles (LB) are compounds that increase the vascular permeability of the tumor under diagnostic ultrasound (US) exposure and enable the effective transport of drugs to tumor cells. The aim of our study was to establish a novel drug delivery technique for chemotherapy and to identify the most effective anticancer drugs for the bubble US-mediated drug delivery system (BUS-DDS) in gynecological cancer treatments. We constructed xenograft models using cervical cancer (HeLa) and uterine endometrial cancer (HEC1B) cell lines. Lipid bubbles were injected i.v., combined with either cisplatin (CDDP), pegylated liposomal doxorubicin (PLD), or bevacizumab, and US was applied to the tumor. We compared the enhanced chemotherapeutic effects of these drugs and determined the optimal drugs for BUS-DDS. Tumor volume reduction of HeLa and HEC1B xenografts following cisplatin treatment was significantly enhanced by BUS-DDS. Both CDDP and PLD significantly enhanced the antitumor effects of BUS-DDS in HeLa tumors; however, volume reduction by BUS-DDS was insignificant when combined with bevacizumab, a humanized anti-vascular endothelial growth factor mAb. The BUS-DDS did not cause any severe adverse events and significantly enhanced the antitumor effects of cytotoxic drugs. The effects of bevacizumab, which were not as dose-dependent as those of the two drugs used prior, were minimal. Our data suggest that BUS-DDS technology might help achieve "reinforced targeting" in the treatment of gynecological cancers.

Enhanced Vascular Permeability by Microbubbles and Ultrasound in Drug Delivery.

Ultrasound and microbubbles, an ultrasound contrast agent, have recently increased attention to developing novel drug delivery systems. Ultrasound exposure can induce mechanical effects derived from microbubbles behaviors such as an expansion, contraction, and collapse depending on ultrasound conditions. These mechanical effects induce several biological effects, including enhancement of vascular permeability. For drug delivery, one promising approach is enhancing vascular permeability using ultrasound and microbubbles, resulting in improved drug transport to targeted tissues. This approach is applied to several tissues and drugs to cure diseases. This review describes the enhancement of vascular permeability by ultrasound and microbubbles and its therapeutic application, including our recent study. We also discuss the current situation of the field and its potential future perspectives.

Model difference in the effect of cilostazol on the development of experimental pulmonary hypertension in rats.

BACKGROUND: Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. METHODS: Fifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. The mean pulmonary artery pressure (mPAP), the right ventricle weight-to-left ventricle + septum weight ratio (RV/LV + S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Levels of the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB proteins in lung tissue were measured using Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. RESULTS: mPAP [35.1 ± 1.7 mmHg (MCT) (n = 9) vs. 16.6 ± 0.7 (control) (n = 9) (P < 0.05); 29.1 ± 1.5 mmHg (CH) (n = 10) vs. 17.5 ± 0.5 (control) (n = 10) (P < 0.05)], RV/LV + S [0.40 ± 0.01 (MCT) (n = 18) vs. 0.24 ± 0.01 (control) (n = 10) (P < 0.05); 0.41 ± 0.03 (CH) (n = 13) vs. 0.27 ± 0.06 (control) (n = 10) (P < 0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1 ± 1.1 mmHg (n = 11) (P < 0.05), RV/LV + S 0.30 ± 0.01 (n = 14) (P < 0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and increased in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats. CONCLUSIONS: We found model differences in the effect of CLZ on PH development. CLZ might exert a preventive effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might depend on the PH etiology.

The Application of the in-Situ Hyperthermia Emission from Acoustic Nanodroplets for Theranostic Dual-Imaging and Antitumor Modalities.

In this study, we have developed a theranostic nanocarrier that can emit heat upon the exposure to ultrasound (US) irradiation as well as the generation of a contrast signal that can be detected with ultrasonography. The prepared acoustic nanodroplets (NDs) made with liquid perfluporopentane (PFPn) had an average size of 197.7 ± 3.6 nm in diameter and were stable in vitro for 60 min. US irradiation at 2 W.cm-2 induced phase change of NDs into bubbles in vitro. On the other hand, the intra-tumor injection of NDs in combination with US irradiation induced thermal emission in situ in B16BL6 melanoma tumor implanted into mice and the emission areas have mostly covered the tumor site. Also, the combination between NDs and US irradiation has inhibited the tumor growth. Under this condition, the heat shock protein (HSP70) in tumor was significantly upregulated after 6 h of the treatment of NDs with US. Thus, we have developed a therapeutic system with multiple theranostic modalities composed of acoustic NDs and US irradiation applicable to the tumor treatment on the external surface of the body.

Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for ultrasound imaging and therapy.

Microbubble formulations have a long history for enhancement of ultrasound (US) imaging and recently also for therapeutic applications. Previously, a series of freeze-dried bubble formulations based on the lipids DSPC and DSPG were developed. Here, we have attempted to scale-up the production process for future more extensive studies. Bubbles were prepared by homogenization of a lipid dispersion in a perfluoropropane atmosphere in a medium size (300-500 mL) homogenizer and then freeze-dried for better storage stability. In total, 300 freeze-dried vials were prepared. The properties of the bubbles were similar to those previously prepared on a lab scale with the difference that they were slightly larger and also had a better stability. The re-entrapped gas concentration after re-constituted freeze-dried bubbles was 9.4 µL/µmol lipid. The re-entrapped rate was 72.3% of fresh bubble before freeze-drying (13.0 µL/µmol lipid). The half-life of US imaging signal of the re-constituted freeze-dried bubbles in water in vitro was shorter than that of the fresh bubbles (2.7 min vs. 3.3 min). A leak of Evans Blue, that binds to albumin, from mouse ear blood vessel was observed after combination of bubble and US irradiation of 1 MHz for 1 min. As a result of bubble vibration by US irradiation, vascular endothelial cell bond opened and Evans Blue leaked. Toxicity of bubble was tested in rats. No toxicity was found after a single injection in the dose range tested. No serious toxicity was seen after repeated injections (one daily injection during 15 days).

Evaluation of the Theranostic Potential of Perfluorohexane-Based Acoustic Nanodroplets.

In this study, we have prepared perfluorohexane (PFH)-based acoustic nanodroplets (PFH-NDs) and evaluated their theranostic characteristics. Nile Red (NR) was incorporated into PFH-NDs as a model of hydrophobic drugs (NR-PFH-NDs). The mean particle diameters of PFH-NDs and NR-PFH-NDs were 205 ± 1.8 nm and 346.3 ± 6 nm, respectively. There was no significant PFH leakage from PFH-NDs during 90 min incubation at 37°C in the presence of 10% rat serum. The in vitro ultrasonography showed that the phase transition of PFH-NDs from liquid droplets to gassed bubbles could be induced by therapeutic low-intensity ultrasound with a frequency of 1 MHz and an intensity of 5 W/cm2. Irradiation of ultrasound in combination with NR-PFH-NDs enhanced uptake of NR in murine adenocarcinoma cells (C26). After intravenous injection of PFH-NDs to mice, PFH gradually disappeared from blood circulation with an elimination half-life of 43.3 min. Intravenous injection of PFH-NDs also resulted in significant contrast enhancement in the mouse carotid artery upon therapeutic low-intensity ultrasound irradiation. These results suggest the potential of PFH-NDs as a novel contrast agent for further theranostic applications.

Development of Dendritic Cell-Based Immunotherapy Targeting Tumor Blood Vessels in a Mouse Model of Lung Metastasis.

Tumor blood vessels supply cancer tissues with oxygen and nutrients, and it was therefore believed that inhibition of angiogenesis would induce tumor regression. In fact, the situation is complicated by the presence of normal blood vessels in cancer tissues such as carcinomas and sarcomas as well as abnormal vessels. Here, we describe the development of a dendritic cell (DC)-based immunotherapy which targets tumor endothelial cells (TECs) rather than normal endothelial cells (ECs) or cancer cells themselves. After density gradient centrifugation, the TEC-rich fraction from lungs invaded by B16 melanoma cells was separated from the endothelial cell (EC)-rich fraction on the basis of positivity for angiotensin-converting enzyme (ACE) activity. Prophylactic vaccination with DCs pulsed with lysates of TECs isolated from lungs with metastases significantly suppressed lung metastasis in this B16/BL6 mouse melanoma model. This suggests that DC-based vaccine therapy targeting TECs in cancers tissue could show promise as an effective therapy for distant metastasis.