Ultrasound targeted microbubble destruction assisted exosomal delivery of miR-21 protects the heart from chemotherapy associated cardiotoxicity.

Chemotherapy related cardiotoxicity is now becoming one of the biggest hurdles for the prognosis of cancer patients. Therapeutically delivering protective small RNAs holds promise for the cardiotoxicity prevention and therapy. However, heart is intrinsically refractory to the nanoparticle-mediated drug delivery. In this study, we found that the exosome-mediated miRNA delivery into the heart could be significantly augmented with the aid of ultrasound targeted microbubble destruction (UTMD). Moreover, we found that UTMD assisted exosomal miR-21 delivery into the heart significantly decreased the cell death, and restored the cardiac function in a doxorubicin induced cardiotoxicity mouse model. Our study here not only provides a promising strategy to protect the heart from the chemotherapy related cardiotoxicity, but also sheds light on gene therapy of other heart diseases.

In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9.

In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.

Maternal exosomes in diabetes contribute to the cardiac development deficiency.

Maternal diabetes mellitus induces an increased risk of congenital heart defects (CHD), however, the exact mechanisms are still not fully illustrated. In this study, diabetic pregnant C57BL/6 mice were induced by injection of streptozotocin before mating. Compared with the control normal mice, diabetic pregnant mice displayed significant changes of the exosomal miRNA contents in the blood, as revealed by RNA-seq analysis. Multiple of these miRNAs were found involved in cardiac development regulation. Moreover, fluorescence labeled exosomes and gold nanoparticles could cross the placenta barrier and infiltrated into the embryonic organs/tissues, including the heart, during embryonic development. Injection of diabetic maternal exosomes strikingly increased the risk of CHD in the normal recipient pregnant mice. Taken together, we could draw the conclusion that maternal exosomes in diabetes could cross the maternal-fetal barrier and contribute to the cardiac development deficiency possibly via miRNAs, providing new insights in CHD prevention and treatment.

Study of the Blood Supply Fraction of the Ascending Aorta and Its Effect in Diagnosing Early Ascending Aortic Atherosclerosis.

OBJECTIVES: To investigate the capacity of blood storage of certain large arteries during diastole, we first studied the ascending aorta by echocardiography. The concept of the blood supply fraction of the ascending aorta was then introduced to evaluate elastic retraction of the ascending aortic wall and determine its role in diagnosing early atherosclerosis of the ascending aorta. METHODS: First, we enrolled 120 healthy volunteers and divided them into 3 groups according to age: 20 to 35 years (B1 group), 36 to 50 years (B2 group), and 51 to 65 years (B3 group); there were 40 volunteers in each group. We used echocardiography to measure the blood supply fraction in each volunteer and compared the results for each group. Then we enrolled 40 patients (51-65 years) with early atherosclerosis of the ascending aorta, measured the blood supply fraction of each, and compared the results with the B3 group. RESULTS: The mean blood supply fractions ± SD in the B1, B2, and B3 groups were 21.75% ± 1.53%, 20.76% ± 1.62%, and 18.44% ± 1.19%, respectively. The fraction in the B3 group was significantly lower than those in the B1 and B2 groups (P < .01). The fraction in the patients with early atherosclerosis was 14.92% ± 1.01%, which was obviously lower than that in the B3 group (P < .01). CONCLUSIONS: The blood supply fraction of the ascending aorta decreases with age, and it could be used as a parameter for diagnosis of early atherosclerosis of the ascending aorta.

Supplementation of Clostridium butyricum Alleviates Vascular Inflammation in Diabetic Mice.

BACKGRUOUND: Gut microbiota is closely related to the occurrence and development of diabetes and affects the prognosis of diabetic complications, and the underlying mechanisms are only partially understood. We aimed to explore the possible link between the gut microbiota and vascular inflammation of diabetic mice. METHODS: The db/db diabetic and wild-type (WT) mice were used in this study. We profiled gut microbiota and examined the and vascular function in both db/db group and WT group. Gut microbiota was analyzed by 16s rRNA sequencing. Vascular function was examined by ultrasonographic hemodynamics and histological staining. Clostridium butyricum (CB) was orally administered to diabetic mice by intragastric gavage every 2 days for 2 consecutive months. Reactive oxygen species (ROS) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by fluorescence microscopy. The mRNA expression of inflammatory cytokines was tested by quantitative polymerase chain reaction. RESULTS: Compared with WT mice, CB abundance was significantly decreased in the gut of db/db mice, together with compromised vascular function and activated inflammation in the arterial tissue. Meanwhile, ROS in the vascular tissue of db/db mice was also significantly increased. Oral administration of CB restored the protective microbiota, and protected the vascular function in the db/db mice via activating the Nrf2/HO-1 pathway. CONCLUSION: This study identified the potential link between decreased CB abundance in gut microbiota and vascular inflammation in diabetes. Therapeutic delivery of CB by gut transplantation alleviates the vascular lesions of diabetes mellitus by activating the Nrf2/HO-1 pathway.

Colon specific delivery of miR-155 inhibitor alleviates estrogen deficiency related phenotype via microbiota remodeling.

Compelling data have indicated menopause-associated increase in cardiovascular disease in women, while the underlying mechanisms remain largely unknown. It is established that changes of intestinal microbiota affect cardiovascular function in the context of metabolic syndrome. We here aimed to explore the possible link between host intestinal function, microbiota, and cardiac function in the ovariectomy (OVX) mouse model. Mice were ovariectomized to induce estrogen-related metabolic syndrome and cardiovascular defect. Microbiota was analyzed by 16s rRNA sequencing. miRNA and mRNA candidates expression were tested by qPCR. Cardiac function was examined by echocardiography. Colon specific delivery of miRNA candidates was achieved by oral gavage of Eudragit S100 functionalized microspheres. In comparison with the sham-operated group, OVX mice showed compromised cardiac function, together with activated inflammation in the visceral adipose tissue and heart. Lactobacillus abundance was significantly decreased in the gut of OVX mice. Meanwhile, miR-155 was mostly upregulated in the intestinal epithelium and thus the feces over other candidates, which in turn decreased Lactobacillus abundance in the intestine when endocytosed. Oral delivery of miR-155 antagonist restored the protective microbiota and thus protected the cardiac function in the OVX mice. This study has established a possible regulatory axis of intestinal miRNAs-microbiota-estrogen deficiency related phenotype in the OVX model. Colon specific delivery of therapeutic miRNAs would possibly restore the microbiota toward protective phenotype in the context of metabolic syndrome.

Exosome-mediated delivery of inflammation-responsive Il-10 mRNA for controlled atherosclerosis treatment.

Rationale: Tailored inflammation control is badly needed for the treatment of kinds of inflammatory diseases, such as atherosclerosis. IL-10 is a potent anti-inflammatory cytokine, while systemic and repeated delivery could cause detrimental side-effects due to immune repression. In this study, we have developed a nano-system to deliver inflammation-responsive Il-10 mRNA preferentially into macrophages for tailored inflammation control. Methods:Il-10 was engineered to harbor a modified HCV-IRES (hepatitis C virus internal ribosome entry site), in which the two miR-122 recognition sites were replaced by two miR-155 recognition sites. The translational responsiveness of the engineered mRNA to miR-155 was tested by Western blot or ELISA. Moreover, the engineered Il-10 mRNA was passively encapsulated into exosomes by forced expression in donor cells. Therapeutic effects on atherosclerosis and the systemic leaky expression effects in vivo of the functionalized exosomes were analyzed in ApoE-/- (Apolipoprotein E-deficient) mice. Results: The engineered IRES-Il-10 mRNA could be translationally activated in cells when miR-155 was forced expressed or in M1 polarized macrophages with endogenous miR-155 induced. In addition, the engineered IRES-Il-10 mRNA, when encapsulated into the exosomes, could be efficiently delivered into macrophages and some other cell types in the plaque in ApoE-/- mice. In the recipient cells of the plaque, the encapsulated Il-10 mRNA was functionally translated into protein, with relatively low leaky in other tissues/organs without obvious inflammation. Consistent with the robust Il-10 induction in the plaque, exosome-based delivery of the engineered Il-10 could alleviate the atherosclerosis in ApoE-/- mice. Conclusion: Our study established a potent platform for controlled inflammation control via exosome-based systemic and repeated delivery of engineered Il-10 mRNA, which could be a promising strategy for atherosclerosis treatment.

Maternal obesity increases the risk of fetal cardiac dysfunction via visceral adipose tissue derived exosomes.

INTRODUCTION: There is a strong association between gestational obesity and fetal cardiac dysfunction, while the exact mechanisms remain largely unknown. The purpose of this study was to investigate the role of exosomes from maternal visceral adipose tissue in abnormal embryonic development in obese pregnancy. METHODS: Female C57BL/6J obese mice were induced by a high-fat diet (containing 60% fat). Fetal cardiac function and morphology were examined by echocardiography and histology. The placenta was extracted for histological examination. miRNA expression in exosomes from the visceral adipose tissue was profiled by RNA-seq. Gene expression of inflammatory factors was analyzed by qPCR. RESULTS: In the obese pregnant mice, there were obvious inflammation and lipid droplets in the placenta. And the fetal cardiac function in obese pregnancy was also compromised. Moreover, injection of the visceral adipose tissue exosomes from the obese mice significantly decreased the fetal cardiac function in the normal lean pregnant mice. Mechanistically, the decreased expression of miR-19b might be responsible for the enhanced inflammation in the placenta. DISCUSSION: Exosomes derived from visceral adipose tissue in obese mice contribute to fetal heart dysfunction, at least partially via affecting the function of the placenta.

Ultrasound Assisted Exosomal Delivery of Tissue Responsive mRNA for Enhanced Efficacy and Minimized Off-Target Effects.

Exosome-mediated nucleic acids delivery has been emerging as a promising strategy for gene therapy. However, the intrinsic off-target effects due to non-specific uptake of exosomes by other tissues remain the big hurdle for clinical application. In this study, we aimed to enhance the efficacy and minimize the off-target effects by simultaneously encapsulating engineered mRNA translationally activated by tissue-specific microRNA (miRNA) and increasing targeted delivery efficiency via ultrasound-targeted microbubble destruction (UTMD). Briefly, the upstream of interest transcript was engineered to harbor an internal ribosome entry site (IRES) modified with two miRNA recognition sites. In vitro reporter experiments revealed that the engineered mRNA could be encapsulated into exosomes and can be translationally activated by corresponding miRNAs in the recipient cells. By a proof-of-principle in vivo experiment, we encapsulated miR-148a (an adipose relatively specific miRNA)-responsive PGC1α mRNA into exosomes and delivered the exosomes into the adipose tissue with the aid of UTMD. Efficient PGC1α translation was activated in the adipose tissue, together with obvious browning induction. Moreover, there was much lower off-target translation of PGC1 α in lungs and other tissues. Taken together, our study establishes a novel adipose-specific exosome delivery strategy to enhance efficacy and minimize off-target effects simultaneously.

Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium.

Rationale: Exosomes are emerging as a promising drug delivery carrier. However, rapid uptake of exosomes by the mononuclear phagocyte system (MPS) remains an obstacle for drug delivery into other targeted organs, including the heart. We hypothesized that prior blocking of uptake of exosomes by the MPS would improve their delivery to the targeted organs. Methods: Exosomes were isolated from the cell culture medium. Fluorescence-labeled exosomes were tracked in vitro and in vivo by fluorescence imaging. The expression of clathrin heavy chain (Cltc), cavolin1, Pak1 and Rhoa, known genes for endocytosis, were profiled in various cell lines and organs by qPCR. The knockdown efficiency of siRNA against Cltc was analyzed by Western blotting. Exosomecontrol and exosomeblocking were constructed by encapsulating isolated exosomes with siControl or siClathrin via electroporation, while exosometherapeutic was constructed by encapsulating isolated exosomes with miR-21a. Doxorubicin-induced cardiotoxicity model was used to verify the therapeutic efficiency of the exosome-based miR-21a delivery by echocardiography. Results: Exosomes were preferentially accumulated in the liver and spleen, mainly due to the presence of abundant macrophages. Besides the well-known phagocytic effect, efficient endocytosis also contributes to the uptake of exosomes by macrophages. Cltc was found to be highly expressed in the macrophages compared with other endocytosis-associated genes. Accordingly, knockdown of Cltc significantly decreased the uptake of exosomes by macrophages in vitro and in vivo. Moreover, prior injection of exosomeblocking strikingly improved the delivery efficiency of exosomes to organs other than spleen and liver. Consistently, compared with the direct injection of exosometherapeutic, prior injection of exosomeblocking produced a much better therapeutic effect on cardiac function in the doxorubicin-induced cardiotoxicity mouse model. Conclusions: Prior blocking of endocytosis of exosomes by macrophages with exosomeblocking successfully and efficiently improves the distribution of following exosometherapeutic in targeted organs, like the heart. The established two-step exosome delivery strategy (blocking the uptake of exosomes first followed by delivery of therapeutic exosomes) would be a promising method for gene therapy.

Fusion protein engineered exosomes for targeted degradation of specific RNAs in lysosomes: a proof-of-concept study.

Therapeutically intervening the function of RNA in vivo remains a big challenge. We here developed an exosome-based strategy to deliver engineered RNA-binding protein for the purpose of recruiting specific RNA to the lysosomes for degradation. As a proof-of-principle study, RNA-binding protein HuR was fused to the C-terminus of Lamp2b, a membrane protein localized in both exosome and lysosome. The fusion protein was able to be incorporated into the exosomes. Moreover, exosomes engineered with Lamp2b-HuR successfully decreased the abundance of RNA targets possibly via lysosome-mediated degradation, especially when the exosomes were acidified. The system was specifically effective in macrophages, which are lysosome enriched and resistant to routine transfection mediated RNAi strategy. In the CCl4-induced liver injury mouse model, we found that delivery of acidified exosomes engineered with Lamp2b-HuR significantly reduced liver fibrosis, together with decreased miR-155 and other inflammatory genes. In summary, the established exosome-based RNA-binding protein delivery strategy, namely "exosome-mediated lysosomal clearance", takes the advantage of exosome in targeted delivery and holds great promise in regulating a set of genes in vivo.

Lactobacillus supplementation prevents cisplatin-induced cardiotoxicity possibly by inflammation inhibition.

PURPOSE: Cardiotoxicity of chemotherapy exerts as the main hurdle for prognosis, while whether cisplatin causes severe cardiotoxicity remains largely unknown. Accumulating evidence reveals that intestinal microbiota functions importantly in nutrient metabolism and cardiovascular function. In this study, we observed the possible cardiotoxicity of cisplatin and explored the possible role of microbiota in the mouse model. METHODS: C57Bl6 mice were treated with 0, 3 or 6 mg/kg cisplatin via i.p. injection, together with or without Lactobacillus supplementation. Cardiac function was analyzed by echocardiography. Gut microbiota was analyzed by 16S RNA sequencing. Gene expression was analyzed by qPCR. The data differences were compared with Graphpad Prism 7.0. RESULTS: In comparison with the control group, 6 mg/kg per week cisplatin treatment for 3 weeks significantly decreased the body weight by about 33% (18.1 ± 2.1 vs 27.2 ± 0.9) and decreased the left ventricular ejection fraction by about 15% (0.57 ± 0.07 vs 0.67 ± 0.04). Together, the gut microbiota was found dramatically changed, manifested as 27% decrease of Firmicutes and increased pathological bacteria. Antibiotics treatment had no obvious beneficial effects on the body weight and cardiac function caused by cisplatin. However, Lactobacillus supplementation significantly increased the body weight and restored cardiac function, together with lower inflammation gene expression. CONCLUSIONS: The study here has established a possible role of microbiota dysbiosis in cisplatin-associated toxic effects, while delivery of Lactobacillus would be beneficial for the cardiac function prevention possibly via inflammation control.

New method for quantifying and correcting underestimated cardiac Doppler blood flow velocities: in vitro and in vivo studies.

This study was aimed to quantify the underestimation of cardiac Doppler measurements and to explore a method for correction. A dual pulse wave (PW)/Doppler tissue imaging (DTI) mode echocardiographic technique was used in the in vitro and in vivo studies. In the in vitro experiment, we have demonstrated how cardiac valvular motion might interfere with blood velocity estimation using conventional Doppler. When examining the participants, we observed that adding valvular annulus velocity to determine the relative velocity between blood and valvular annulus would result in an increment of 9.3 ± 1.3 cm/s and 6.3 ± 0.9 cm/s for aortic and pulmonary blood flow, 12.8 ± 1.9 cm/s and 8.9 ± 1.4 cm/s for mitral E and A wave, 12.9 ± 1.8 cm/s and 10.2 ± 2.4 cm/s for tricuspid E and A wave. The underestimations of the Doppler measurements markedly influence the hemodynamic parameters commonly used in the clinical practices and researches. This study provides a quantitative method for the correction and would make the Doppler measurement accurate.