Inhibitory effect on subretinal fibrosis by anti-placental growth factor treatment in a laser-induced choroidal neovascularization model in mice

AIM:To investigate whether anti-placental growth factor(PGF) can inhibit subretinal fibrosis and whether this effect is mediated by the inhibitory effect of PGF on epithelial-mesenchymal transition(EMT) of retinal pigment epithelial(RPE) cells.METHODS:Subretinal fibrosis model was established in laser induced choroidal neovascularization(CNV) mice on day 21 after laser photocoagulation.Immunofluorescence staining(IFS) of cryosections and enzyme-linked immunosorbent assay(ELISA) were used to detect the expression of PGF.IFS of whole choroidal flat-mounts was used to detect the degree of subretinal fibrosis.IFS of cryosections and ELISA were used to detect the expression of EMT related indicators in subretinal fibrosis lesions.RESULTS:The expression of PGF protein in subretinal fibrosis lesions was significantly up-regulated(P<0.05),and mainly co-stained with pan-cytokeratin labeled RPE cells.Intravitreal injection of anti-PGF neutralizing antibody reduced the area of subretinal fibrosis and the ratio of fibrotic/angiogenic area significantly at the concentrations of 0.25,0.5,1.0,and 2.0 μg/μL(all P<0.05).The expression of E-cadherin in the local RPE cells decreased,while α-SMA increased significantly in subretinal fibrosis lesions,and the application of anti-PGF neutralizing antibody could reverse these changes(P<0.05).CONCLUSION:The expression of PGF is up-regulated in the lesion site of subretinal fibrosis and mainly expressed in RPE cells.Intravitreal injection of anti-PGF neutralizing antibody can significantly inhibit the degree of subretinal fibrosis in CNV mice,and this effect may be mediated by the inhibition of PGF on EMT of RPE cells.

Analysis of Placental Growth Factor in Placentas of Normal Pregnant Women and Women with Hypertensive Disorders of Pregnancy

To investigate the expressions of placental growth factor （PLGF） in placenta with hypertensive disorders of pregnancy （ HDP）, 45 women with HDP and 20 normally pregnant women were studied. Among 45 women with HDP, there were 23 cases of severe preeclampsia and one case of eclampsia. The location and level of PLGF proteins was determined by immunohistochemistry and Western blot. The expression of PLGF mRNA in placenta was assessed by reverse transcriptionalpolymerase chain reaction （RT-PCR）. The results showed that： （1） The distribution of PLGF in placenta with HDP was similar to normal one, which was mainly in the cytoplasm of villous syncytiotrophoblast and villous stroma; （2） The expression of PLGF protein was significantly decreased in placentas with mild and severe preeclampsia compared to the normal ones （0.3±0.4 vs 0.6± 0.4, 0.2±0.5 vs 0. 6±0. 4, P〈0.01）. There were no differences between the gestational hypertension placenta and normal one （0.5±0.6 vs 0. 6±0. 4, P〉0.05） ; （3） The transcription levels of the PLGF mRNA in placentas with preeclampsia were significantly lower than in normal groups （3.33±0.39 vs 4.87±0. 60, 1.97±0.29 vs 4.87±0.60, P〈0.01）, and no differences were found between the gestational hypertension placenta and normal groups. These findings suggest that the abnormal expression of PLGF in placentas is related to the pathogenesis of HDP.

Effect of Compound Danshen Injection Combined with Labetalol on Liver Function and Placental Growth Factor in Patients with Eclampsia

Objective: To explore the effect of Compound Danshen Injection combined with Labetalol on liver function and placental growth factor in patients with eclampsia. Methods: Seventy patients with eclampsia who were treated in the Hospital from February 2017 to February 2019 were enrolled. The patients were divided into two groups according to the random number table, with 35 cases in each group. The Observation group was treated with Labetalol, and the combined therapy group was treated with Compound Danshen Injection combined with Labetalol. The liver function [alanine aminotransferase, aspartate aminotransferase, total protein, and albumin], hemorheology indicators, placental growth factor and serum insulin-like growth factor-1 and clinical indicators in the two groups were analyzed. Results: After treatment, the levels of alanine aminotransferase and aspartate aminotransferase in the combined therapy group were significantly lower than those in the Observation group. The levels of total protein and albumin in the combined therapy group were significantly higher than those in the Observation group (P<0.05). After treatment, the high-cut and low-cut whole blood viscosity, plasma viscosity and erythrocyte rigidity index in the combined therapy group were significantly lower than the Observation group, and the difference was statistically significant (P<0.05). After treatment, the levels of PLGF and IGF-1 in the combined therapy group were significantly higher than those in the Observation group, and the difference was statistically significant (P<0.05). After treatment, the gestational age, neonatal weight index, placental weight and neonatal 1 min Apgar score in the combined therapy group were significantly higher than the Observation group, and the difference was statistically significant (P<0.05). Conclusion: For patients with eclampsia, Compound Danshen Injection combined with Labetalol is with great safety, which can help stabilize their condition, and improve their liver function and placental growth factor status.

Placental growth factor expression is reversed by anti-vascular endothelial growth factor therapy under hypoxic conditions

Background:Clinical trials have revealed that the antivascular endothelial growth factor(VEGF)therapies are effective in retinopathy of prematurity(ROP).But the low level of VEGF was necessary as a survival signal in healthy conditions,and endogenous placental growth factor(PIGF)is redundant for development.The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the infl uence of anti-VEGF therapy on PIGF.Methods:CoCl2-induced hypoxic human umbilical vein endothelial cells(HUVECs)were used for an in vitro study,and oxygen-induced retinopathy(OIR)mice models were used for an in vivo study.The expression patterns of PIGF under hypoxic conditions and the infl uence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction(RTPCR).The retinal avascular areas and neovascularization(NV)areas of anti-VEGF,anti-PIGF and combination treatments were calculated.Retina PIGF concentration was evaluated by ELISA after treatment.The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies.Results:PIGF mRNA expression was reduced by hypoxia in OIR mice,in HUVECs under hypoxia and anti-VEGF treatment.However,PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia.Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions,but it stimulated cell proliferation and migration under hypoxia.Anti-PIGF treatment was effective for neovascular tufts in OIR mice(P<0.05).Conclusion:The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

Placenta Growth Factor and Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia and Normotensive Pregnant Nigerian Women

Background: Preeclampsia (PE) is still one of the leading causes of maternal/perinatal morbidity/mortality in Nigeria. Imbalance between placenta growth factor (PLGF) and soluble fms-like tyrosine kinase 1 (sFlt1) has been reportedly present both before and after the manifestation of PE;however, Nigerian data regarding these angiogenesis-related substances are lacking. We here attempted to determine the maternal serum level of PLGF and sFlt1 and sFlt1/PLGF ratio in PE vs. non-PE women in Lagos State University Teaching Hospital, Nigeria. Methods: An observational cross-sectional study was made on 75 women with PE and 75 age-gestational-age matched women without PE, as case and control, respectively. Levels of sFlt-1, PIGF and the sFlt-1: PIGF ratio was compared between the two. Results: Serum levels of Flt-1 and sFlt1/PIGF ratio were significantly higher in PE patients (6581.86 ± 865.75, and 146.42 ± 92.43) than in the normotensive control (4584.52 ± 1479.6 and 11.60 ± 6.42). PIGF was significantly lower in PE patients (70.14 ± 51.03) than the normotensives (494.06 ± 475.8). There were positive and negative correlations between the sFlt-1 and PLGF respectively and mean arterial blood pressure. Conclusion: Serum sFlt-1, sFlt1/PIGF ratio was significantly higher and PIGF levels were significantly lower in PE than normotensive control in Nigerian population.

Serum Soluble Vascular Endothelial Growth Factor Receptor 1 as a Potential Biomarker of Hepatopulmonary Syndrome

Background and Aims:The results of basic research implicate the vascular endothelial growth factor(VEGF)family as a potential target of hepatopulmonary syndrome(HPS).However,the negative results of anti-angiogenetic therapy in clinical studies have highlighted the need for markers for HPS.Therefore,we aimed to determine whether VEGF family members and their receptors can be potential biomarkers for HPS through clinical and experimental studies.Methods:Clinically,patients with chronic liver disease from two medical centers were enrolled and examined for HPS.Patients were divided into HPS,intrapulmonary vascular dilation[positive contrast-enhanced echocardiography(CEE)and normal oxygenation]and CEE-negative groups.Baseline information and perioperative clinical data were compared between HPS and non-HPS patients.Serum levels of VEGF family members and their receptors were measured.In parallel,HPS rats were established by common bile duct ligation.Liver,lung and serum samples were collected for the evaluation of pathophysiologic changes,as well as the expression levels of the above factors.Results:In HPS rats,all VEGF family members and their receptors underwent significant changes;however,only soluble VEGFR1(sFlt-1)and the sFlt-1/placental growth factor(PLGF)ratio were changed in almost the same manner as those in HPS patients.Furthermore,through feature selection and internal and external validation,sFlt-1 and the sFlt-1/PLGF ratio were identified as the most important variables to distinguish HPS from non-HPS patients.Conclusions:Our results from animal and human studies indicate that sFlt-1 and the sFlt-1/PLGF ratio in serum are potential markers for HPS.

Clinical efficacy and changes of serum VEGF-A,VEGF-B,and PLGF after conbercept treating neovascular agerelated macular degeneration

AIM:To evaluate the clinical efficacy and systemic safety profile of conbercept in clinical practice on vascular endothelial growth factor(VEGF)-A,VEGF-B,and placental growth factor(PLGF)levels after intravitreal injections for the neovascular age-related macular degeneration(AMD).METHODS:Thir ty-five patients(35 eyes)with neovascular AMD received intravitreal injections of conbercept treatment with pro re nata protocol.Bestcorrected visual acuity(BCVA)and central retinal thickness(CRT)were detected before the intravitreal injection and at 1,3,and 12mo after conbercept treatment.The levels of serum VEGF-A,VEGF-B,and PLGF were measured by enzyme-linked immunosorbent assay before the injection and 1 and 12mo after conbercept treatments.RESULTS:At baseline,the mean BCVA score was 39.89±14.64 letters.The mean BCVA scores were 51.03±15.78,56.71±14.38,and 52.49±10.16 letters at 1,3,and 12mo after conbercept treatment,and the BCVA improvements were all significant,respectively(P<0.05).At baseline,the mean CRT was 436.7±141.9μm.At 1,3,and 12mo after conbercept treatment,the mean CRT values were 335.1±147.8,301.1±116.5,and 312.2±98.22μm,and the CRT improvements were all significant,respectively(P<0.05).At baseline,1 and 12mo after conbercept treatment,the mean levels of serum VEGF-A were 1013.8±454.3,953.1±426.4,and 981.5±471.7 pg/mL,the mean levels of serum VEGF-B were 46.93±24.76,42.99±19.16,and 45.32±18.76 pg/mL,the mean levels of serum PLGF at these points were 251.7±154.9,241.3±166.7,and 245.6±147.2 pg/mL,respectively.Compared with the baseline,the levels of serum VEGF-A,VEGF-B,and PLGF did not significantly change at 1 and 12mo after conbercept treatment,respectively(P>0.05).CONCLUSION:Conbercept intravitreal injection leads to BCVA and CRT improvement,however,it does not significantly affect systemic serum VEGF-A,VEGF-B,and PLGF levels at 1 and 12mo after intravitreal injection treating neovascular AMD.

Maternal Vascular Dysfunction in Congenital Heart Defects

Background:Research on fetal congenital heart defect(CHD)mostly focuses on etiology and mechanisms.However,studies on maternal complications or pathophysiology are limited.Our objective was to determine whether vascular dysfunction exists in pregnant women carrying a fetus with congenital heart defects.Methods:We conducted a case-control study.27 cases of pregnant women carrying a fetus with major CHD admitted to our hospital for delivery between April 2021 and August 2022 were selected.Every case was matched with about 2 pregnant complication-free controls without fetal abnormalities.The proangiogenic and anti-angiogenic factors and pregnancy outcomes were compared.Results:The proangiogenic factors include vascular endothelial growth factor(VEGF)and placental growth factor(PlGF).The anti-angiogenic factors involve soluble fms-like tyrosine kinase 1(sFlt-1)and soluble endoglin(sEng).No differences were found in maternal plasma concentrations of PlGF,VEGF,and sFlt-1 between case-control groups when analyzed at 36 weeks≤gestational age(GA)<39 weeks and 39 weeks≤GA≤41 weeks.The concentrations of sEng in maternal plasma in the fetal CHD group were significantly higher than those in the control group:0.60(0.77)vs.0.32(0.26)ng/ml at 36 weeks≤GA<39 weeks,p=0.001 and 0.75(0.55)vs.0.28(0.27)ng/ml at 39 weeks≤GA≤41 weeks,p<0.001.Conclusion:Vascular dysfunction exists in pregnant women with fetal congenital heart defects,manifesting significantly elevated sEng concentration at delivery.

Apolipoprotein A1 suppresses the hypoxia-induced angiogenesis of human retinal endothelial cells by targeting PlGF

AIM:To investigate the anti-angiogenic effect of apolipoprotein A1(apoA1)on primary human retinal vascular endothelial cells(HRECs)and explore the possible mechanism.METHODS:The primary HRECs were transfected with apoA1-GFP recombinant lentiviral and were compared with cells undergoing transfection with empty lentiviral vectors.Hypoxia chambers were used to simulate the anoxic environment of cells under pathological condition.The concentrations of secreted vascular endothelial growth factor(VEGF)and placental growth factor(PlGF)were measured by enzyme-linked immunosorbent assay(ELISA).Cell migration ability was detected by wound healing assay.The sprouting of HRECs was determined by tube formation assay.The protein levels of extracellular signal regulated kinase 1/2(ERK1/2)and phosphor ylated ERK1/2(p-ERK1/2)were measured by Western blot.RESULTS:Overexpressed apoA1 in hypoxia-induced HRECs significantly suppressed PlGF(0.67±0.10 folds,P=0.007).Overexpressed apoA1 also attenuated hypoxiainduced cell migration(0.32±0.11 folds,P<0.0001),tube formation(0.66±0.01 folds,P<0.0001)and the phosphorylation levels of ERK(0.6±0.11 folds,P=0.025).Pretreatment of mitogen-activated protein kinase kinase(MEK)inhibitor(U0126)further reduced the PlGF and angiogenesis in hypoxia-induced HRECs.CONCLUSION:ApoA 1 inhibits the angiogenesis at least in part by inactivating ERK1/2 in hypoxia-induced HRECs.Moreover,apoA1 suppresses the PlGF expression,which selectively associated with pathological angiogenesis.

Systematic development of ionizable lipid nanoparticles for placental mRNA delivery using a design of experiments approach

Ionizable lipid nanoparticles(LNPs)have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies.LNPs enhance mRNA stability,circulation time,cellular uptake,and preferential delivery to specific tissues compared to mRNA with no carrier platform.However,LNPs are only in the beginning stages of development for safe and effective mRNA delivery to the placenta to treat placental dysfunction.Here,we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity.We conducted a Design of Experiments to explore how LNP composition,including the type and molar ratio of each lipid component,drives trophoblast and placental delivery.Our data revealed that utilizing C12-200 as the ionizable lipid and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE)as the phospholipid in the LNP design yields high transfection efficiency in vitro.Analysis of lipid molar composition as a design parameter in LNPs displayed a strong correlation between apparent pKa and poly(ethylene)glycol(PEG)content,as a reduction in PEG molar amount increases apparent pKa.Further,we present one LNP platform that exhibits the highest delivery of placental growth factor mRNA to the placenta in pregnant mice,resulting in synthesis and secretion of a potentially therapeutic protein.Lastly,our high-performing LNPs have no toxicity to both the pregnant mice and fetuses.Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta,and our top LNP formulations may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

A novel model of retinopathy of prematurity in normobaric hyperoxic conditions

AIM：To examine changes in retinal vasculature after treatment with different oxygen concentrations from common retinopathy of prematurity（ROP） models and to determine a novel and practical ROP model.METHODS：A sample of 14 newborn Sprague-Dawley rats was used.The study group（n=7） was exposed to95%oxygen for 4h per day followed by normoxic laboratory conditions for 20 h.This cycle was repeated for 14 d.The control group（n=7） was subjected to normobaric normoxic conditions.On postnatal day 14（P14）,the two groups were placed in room air for 7d.On P21,the two groups were examined using indirect ophthalmoscopy.All eyes were enucleated for immunofluorescence（IF） staining of the vasculature of retinas and analysis of vascular endothelial growth factor（VEGF）,hypoxia inducible factor-1 alpha（HIF-1α）,placental growth factor（PLGF） in vitreous humor,and then the rats were sacrificed by decapitation.All procedures were repeated using another litter of 14 pups.RESULTS：In the study group and under normobaric hyperoxic conditions,retinal neovascularization and peripheral avascular retina were determined in 85%of the rats through indirect ophthalmoscopic examination.Also IF staining of retina of the study group showed retarded peripheral vascular growth.The difference between the two groups for VEGF,HIF-1α and PLGF concentrations of vitreous humor was statistically significant（P=0.003,0.007,0.027 respectively）.CONCLUSION：Fluctuating oxygen concentrations are primarily responsible for retinal neovascularization.Our new ROP model is practical and applicable for all retinal neovascularization studies,considering the laboratory procedures.

Diagnostic Value of Circulating PIGF in Combination with Flt-1 in Early Cervical Cancer

The utility of placental growth factor(PIGF)and its receptor VEGFR-1(Flt-1)as biomarkers for cervical cancer has not been clarified yet.To address this issue,we investigated the levels of soluble PIGF(sPIGF)and soluble Flt-1(sFlt-1)in the serum from patients with early cervical cancer,cervical intraepithelial neoplasia(CIN)and controls in this study.sPIGF and sFlt-1 were detected in 44 preoperative patients with cervical cancer,18 cases with CIN,and 20 controls by ELISA.It was found that both sPIGF and sFlt-1 were significantly increased in the cervical cancer group as compared with those in CIN and control groups.sPIGF presented a high diagnostic ability of cervical cancer,with a sensitivity of 61.36%and a specifcity of 89.47%;and sFlt-1 with a sensitivity of 50.00%and a specifcity of 92.I 1%。Importantly,the combined use of sPIGF and sFlt-1 could increase the diagnostic rate of cervical cancer,with a sensitivity of 70.45%and a specificity of 92.11%.These results indicated that both sPIGF and sFlt-1 in circulation can serve as possible valuable diagnostic biomarkers for cervical cancer,and the combined use of them can be more valuable to diagnose the patients with early cervical cancer.

Screening and Prevention of Preeclampsia

Preeclampsia(PE)is a multisystem disorder of pregnancy classically characterized by hypertension with significant proteinuria after 20 weeks'gestation.This disorder is one of the leading causes of maternal and perinatal morbidity and mortality.PE can be subdivided into preterm PE(with delivery at<37 weeks'gestation)and term PE(with delivery at≥37 weeks'gestation).Preterm PE is associated with a higher risk of adverse maternal and perinatal outcomes than term PE.Traditional method of screening as recommended by professional guidelines has limited predictive performance and therefore should be updated to reflect recent scientific evidence that the target of screening should be preterm PE,the best way to identify the high-risk group is the Bayes-based method that combines maternal risk factors and biomarkers,the threshold should be set at screen positive rate of 10%,aspirin should be started before 16 weeks'gestation,and the daily dose should be higher than 100mg.