Mechanical Study Of Maternal Inflammatory Exposure Induced Adult Offspring Cardiac Fibrosis And Effects Of BMP10 On Cardiac Fibrosis

Cardiac fibrosis,a common pathophysiologic change of most cardiac diseases,is significantly associated with adverse outcome.Despite the broad range of triggers,the progression of heart failure after the initial injury often follows a common pathway:cardiac injury or stress gives rise to cardiomyocyte death,which in turn gives rise to adverse cardiac remodeling(e.g.,the recruitment and activation of fibroblasts and excessive deposition of extracellular matrix)and ultimately leads to the impairment of cardiac contractile function.According to the newly released report of Report on Cardiovascular Diseases in China2018 by National Center for Cardiovascular Diseases of China,the total number of people with cardiovascular disease in China is approximately 290 million.Cardiovascular diseases,whose related death accounted for more than 43%of all death in rural and urban area of China,is the leading curse of death of China,higher than tumor and any other diseases.Even the primary and secondary prevention were applied widely,it is estimated that the prevalence of cardiovascular diseases would still be on the rising in the up coming decade,suggesting hidden critical etiology of cardiovascular diseases was uncovered.Substantial clinical epidemiological observations have suggested that adverse maternal inflammatory exposure is closely related with the adult onset of cardiovascular diseases.Our research group have proposed a hypothesis that disease states in cardiovascular system may origin from prenatal inflammatory exposure.And we have proved that offspring from prenatal inflammatory exposure can develop hypertension and cardiac remodeling in elder offspring rats.Further mechanical studies revealed that the activation of p38 and interaction between ROS-p38MAPK induced by prenatal inflammatory exposure sensitize offspring to cardiac injury.Our research results have profoundly changed the time window for prevention and treatment of cardiovascular diseases and helps in lower-down the onset of cardiovascular diseases worldwide.However,the underlying mechanism of maternal inflammatory exposure induced adult offspring cardiac fibrosis remains exclusively unknown.BMP10 is a peptide growth factor belonging to the TGF-?superfamily.Previous research of our research group has found than BMP10 is critical in maintaining normal embryonic cardiac development and perinatal-to-postnatal cardiac hypertrophic growth.However,whether BMP10 is involved in the pathogenesis of cardiac fibrosis is largely unknown.The present project was designed to determine novel etiology and means of treatment of cardiac fibrosis by exploring the mechanism of maternal inflammatory exposure induced adult offspring cardiac fibrosis and effects of BMP10 injection on cardiac fibrosis.The present study provides new targets for cardiac fibrosis prevention and treatment.Methods and results:1.Mechanical study of maternal inflammatory exposure induced adult offspring cardiac fibrosis1.1 Altered immune pathways in the offspring-p LPS hearts.To determine the underlying changes induced by prenatal LPS exposure,we performed transcriptomic analysis of 4-week-old,sex-matched offspring-p LPS hearts and offspring-p Saline control hearts.Surprisingly,significantly altered expression was observed for a total of 46 genes,among which 15 genes exhibited up-regulated expression and 31 genes exhibited down-regulated expression,suggesting that the maternal inflammatory exposure only targeted a specific set of pathways in the fetus.Using global canonical pathway analysis with the Ingenuity Pathway Analysis(IPA)software package,type I IFN(IFN-?/?)signaling was found the most significantly impaired pathway in the young offspring-p LPS hearts compared to the offspring-p Saline control hearts.IPA revealed that 2 of the top-ranked networks were associated with cell movement of myeloid cells,viral infection,inflammation of organ and a hypersensitivity reaction in the young offspring-p LPS hearts,which further indicated that prenatal inflammatory treatment caused an enduring hyperinflammatory response in the offspring hearts.1.2 iNKT activation at the MFI and a surge of inflammatory cytokines in fetusWe tested whether iNKTs at the MFI were activated with low-dose LPS by analyzing the surface markers of iNKTs(CD45⁺CD19^-CD3⁺TCR?⁺m CD1d tetramer⁺)in the decidua,placenta,and uterus.Maternal LPS exposure resulted in significant iNKT accumulation in the uterus at 48 hours post LPS exposure and in the decidua and placenta at 72 hours post LPS exposure.Interestingly,CD44 and CD69 double-positive iNKTs(activated iNKTs)were significantly enriched in the decidua and placenta at 72 hours post maternal LPS exposure compared to the same time point in controls.We further analyzed several iNKT-secreted cytokines in the maternal serum and fetal amniotic fluid samples following maternal LPS exposure from the 2-hour time point to the 96-hour time point,which included IL-6,IL-22,and TNF-?.While maternal immediate immune response was expectedly peaked at 2-hour time window post maternal LPS exposure as indicated by a rapid rising of IL-6,IL-22,and TNF-?in maternal serum samples,we found a unique cytokine surge pattern in amniotic fluid,in which TNF-?and IL-22 were peaked at 72 hours post LPS exposure.Unexpectedly,IL-6 level seemed only associated with immediate response,suggesting that IL-6 may not be the key player in this system.We administrated pregnant female mice an iNKT specific activator,?-Galactosylceramide(?-Gal/Cer,3?g/mouse).We analyzed TNF-?,IL-22,and IL-6 levels in the amniotic fluid and found that their pattern of surge were similar to that in maternal LPS exposed amniotic fluid samples.1.3 Enhanced infiltration of monocyte-derived macrophages in the offspring heartsWe tested macrophage at the fetal stage,the fetus-p LPS hearts at 18.5 dpc also had an increased level of monocyte-derived macrophage.MCP-1 levels were significantly upregulated in the offspring-p LPS hearts.Significantly increased level of infiltrated macrophages and monocytes in the offspring-p LPS hearts were observed.However,cardiac DC numbers were unchanged in the offspring-p LPS hearts compared to offspring-p Saline hearts.The blockage of iNKT activation completely suppressed the increase of monocyte-derived macrophages in offspring hearts.?-Gal/Cer treatment was able to significantly induce cardiac macrophage infiltration in the offspring hearts.1.4 Progressive NLRP3 activation in offspring-p LPS heartsWe further analyzed the NLRP3 and NF-?B pathways in 1-,2-and 4-week-old hearts.Interestingly,we did not observe activation of the NLRP3 inflammasome in the 1-and 2-week-old offspring-p LPS hearts but found significant activation of the NLRP3inflammasome in the 4-week-old offspring-p LPS hearts compared to the offspring-p Saline control hearts.Abnormally elevated levels of IL-6,IFN-?and TNF-?but not IL-17A were found in the offspring-p LPS hearts at 4 weeks of age when compared to the age-matched offspring-p Saline control hearts.IL-10 and IL-1?were not detectable in either the offspring-p LPS or offspring-p Saline hearts using this assay.1.5 Maternal inflammatory exposure predisposes the heart to injuryTo validate whether offspring-p LPS mice were predisposed to cardiac injury,we stimulated the mice with isoproterenol(ISO;5?g/gram).Strikingly,severe immune cell infiltration was observed in the ISO-treated offspring-p LPS hearts compared to the ISO-treated offspring-p Saline control hearts.Additional immunobiological and immunofluorescence analyses further confirmed dramatically increased levels of collagen deposition and?-smooth muscle actin(?-SMA)expression in the ISO-treated offspring-p LPS hearts.To further confirm this conclusion,RNA-Seq based transcriptome analysis was performed.Pathways involved in inflammation(e.g.,the Th1 pathway and IFN signaling)and cardiac hypertrophy were also significantly enhanced in the offspring-p LPS hearts compared to the offspring-p Saline control hearts by global canonical pathway analysis.1.6 iNKT activation at maternal fetal interface drives prenatal LPS exposure induced cardiac fibrosis predispositionTo confirm this hypersensitivity to cardiac injury in offspring-p LPS hearts was caused by maternal iNKT activation,we analyzed the offspring-p Gal/Cer hearts similarly treated with ISO.Histological analyses revealed that the ISO treated offspring-p Gal/Cer hearts presented a much higher level of collagen deposition than that in the ISO treated offspring-p DMSO control hearts.Conversely,we also compared the ISO treated offspring-p LPS(CD1d^-/-)and ISO treated offspring-p Saline(CD1d^-/-)control hearts,and we found that ISO treated offspring-p LPS(CD1d^-/-)hearts had greatly reduced cardiac fibrosis to a comparable level of ISO treated offspring-p Saline(CD1d^-/-)control hearts2 Effects of BMP10 on cardiac fibrosis2.1 Assessment of cardiac function in?MHC-Bmp10 mice in response to prolonged isoproterenol treatmentIn an attempt to determine whether elevated levels of Bmp10 expression impacted pathological hypertrophic growth induced by the?-adrenergic agonist ISO,we performed a series of morphological and histological analyses on the ISO-treated?MHC-Bmp10(TG)and nontransgenic(NTG)littermate hearts and compared them to age-matched saline-treated?MHC-Bmp10 and nontransgenic littermate control hearts.Our current data demonstrated that cardiac Bmp10 overexpression dramatically reduced the level of cardiac fibrosis.2.2 De novo activation of BMP10 in postnatal heartsAlthough the above data suggested that BMP10 was an important cardioprotective molecule,it was possible that the apparent cardioprotective phenotype could simply be a secondary phenomenon resulting from smaller cardiomyocytes.To circumvent this problem,we generated an inducible Bmp10 transgenic mouse model(?MHC-e GFP^f-Bmp10)(Figure2A).This model used a floxed-e GFP-stop cassette(e GFP^f)that was placed between the mouse?MHC promoter and a c DNA fragment encoding the full-length mouse Bmp10.We confirmed the activation of Bmp10 expression in?MHC-e GFP^f-Bmp10 by crossing?MHC-Mer Cre Mer mice.Tamoxifen treatment resulted in the activation of Bmp10 expression in?MHC-e GFP^f-Bmp10:?MHC-Mer Cre Mer hearts,as evidenced by q RT-PCR.2.3 De novo Bmp10 induction improves cardiac function in response to isoproterenol infusionTo determine if de novo Bmp10 expression in postnatal hearts exerted cardioprotective activity,we subjected 2-month-old?MHC-e GFP^f-Bmp10:?MHC-Mer Cre Mer mice(with or without tamoxifen induction),as well as nontransgenic mice,to ISO infusion for 7 days.Our results revealed that Bmp10 had strong cardioprotective activity in cardiac fibrosis.The administration of rh BMP10 effectively preserved cardiac function and significantly reduced the cardiac fibrosis induced by prolonged ISO treatment.2.4 Generation of recombinant human BMP10(rh BMP10)protein.To further conform the cardiac protective role of BMP10 in ISO induced cardiac fibrosis,we generated human BMP10 protein in a bio-engineering drug manufacture manner.The bioactivity of BMP10 were evaluated though a luciferase assay in C2C12 cells.And the cardiac protective effects of rh BMP10 in ISO induced cardiac fibrosis were further conformed in two different mouse strains.2.5 Altered SMAD-and STAT3-mediated signaling pathways in BMP10 transgenic heartsWe evaluated the transcriptomes between?MHC-Bmp10 transgenic and nontransgenic littermate hearts and found a total of 402 genes to be differentially expressed between?MHC-Bmp10 transgenic hearts and NTG control hearts and then subjected them to Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)analysis.The results have revealed that TGF?/BMP and its mediated pathways were the top affected signaling pathways in Bmp10 transgenic hearts.Ingenuity Pathway Analysis also revealed that TGF?/BMP and STAT3 signal pathway may involved in the cardiac protective role of BMP10.2.6 Confirmation of the role of BMP10 in activating both SMAD and STAT3 signalingWe used Western blot to analyze the potential activation of STAT3 as well as other noncanonical signaling pathways downstream of TGF?/BMPs.Consistent with IPA analysis,as shown in Figure 5A,p Y-STAT3 was upregulated in BMP10 transgenic hearts.To determine if the activation of pY-STAT3 was a specific function of BMP10,we treated P19cells with rh BMP10 in vitro.The result suggested STAT3 activation was a direct,specific,and immediate response to BMP10 stimulation and was likely an important effector in the BMP10 signaling pathway.2.7 STAT3 deficiency reduces Bmp10-mediated cardioprotectionWe tested whether STAT3 was essential to BMP10-mediated cardioprotection by administrating rh BMP10 to STAT3 cardiomyocyte-specific knockout(STAT3cko)mice.The resulted myocardial STAT3 was essential for BMP10-mediated cardiomyocyte survival while the reduced level of collagen deposition in BMP10-treated hearts was likely independent of myocardial STAT3.2.8 BMP10-mediated inhibition of excessive extracellular matrix depositionTo test whether BMP10 pathway was involved in the inhibition of cardiac fibroblast activity,we isolated adult cardiac fibroblasts for series in vitro experiments.³H-proline incorporation assay revealed rh BMP10 was able to effectively inhibit the production of collagen.Furthermore,q RT-PCR confirmed that both Col1a1 and Col3a1 transcripts were significantly reduced by rh BMP10 treatment.miRNA array revealed miR29,miR30,miR1,miR23,and miR199 were significantly upregulated,and miR34 was significantly downregulated.Among these,miR29c was the most elevated miRNA(over a 3-fold increase,p<0.001).anti-miRs of miR29 significantly reduced BMP10-mediated downregulation of Col1a1 and Col3a1.Conclusions:1.Progressive increase in infiltrated CCR2⁺macrophage and proinflammatory cytokines release in the heart underlined the hypersensitivity of offspring-p LPS hearts to chronic and/or acute cardiac injuries.2.Maternal inflammatory exposure triggered an iNKT activation at MFI,which likely mediated a cytokine surge in fetuses and led to altered cardiac innate immunity3.maternal iNKT knockout can reverse the hypersensitivity of offspring-p LPS hearts to chronic and/or acute cardiac injuries.4.Recombinant human BMP10 protein were generated and purified.5.BMP10 protects heart function in response to ISO pump induced cardiac fibrosis and heart failure via regulating SMAD1/5/8 signaling,STAT3 signaling and miRNA expression.