Angiogenic Profile Research Articles

Angiogenic profiles in in-vitro fertilization/embryo transfer (IVF/ET) treatments: relationship to hypertensive complications in IVF-conceived pregnancies

Angiogenic profiles in in-vitro fertilization/embryo transfer (IVF/ET) treatments: relationship to hypertensive complications in IVF-conceived pregnancies

68 sFlt-1, PlGF and VEGF in differential diagnosis between preeclampsia and systemic lupus nephritis

Systemic lupus erythematosus (SLE) mainly affects young women and pregnancy in these patients has significant morbidity and mortality. Some features of SLE activity, specifically nephritis, are difficult to differentiate from preeclampsia (PE) in clinical practice. It has been proposed the use of angiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and antiangiogenic factors, as soluble Fms-like tyrosine kinase-1 (sFlt-1), for the differential diagnosis between these two conditions, however there are no available data in the literature about these cytokines in pregnant patients with SLE.Objectives:The objective of this study was to evaluate whether there are differences between serum levels of VEGF, PlGF and sFlt-1 in pregnant women with SLE and active disease, inactive disease or preeclampsia. 38 women with SLE (according to American College of Rheumatology classification criteria) followed at a high risk prenatal care were included. They had no other autoimmune disease diagnosed and were divided according to disease activity and presence of PE. Blood collection was performed at third trimester of pregnancy, within 3 weeks of delivery, and frozen for subsequent blinded analysis through ELISA method. According to according to SLEPDAI (Systemic Lupus Erythematosus Pregnancy Disease Activity Index), 23 patients had inactive SLE (group 1),8 had active lupus nephritis (group2) and 7 had PE (group3). The mean gestational age of blood collection was 36.4 weeks for group 1, 35.5 for group 2 and 35.8 for group 3, without statistical difference between groups. The mean values of VEGF, PlGF and sFlt-1 of all groups are described on Table 1. Considering VEGF and PlGF, patients with SLE and PE had significantly lower mean serum levels than SLE patients without PE, while sFlt-1 was significantly higher in patients with PE compared to inactive SLE and SLE nephritis. The ratio sFlt-1/PlGF was also significantly higher in patients of group 3(PE) compared to other patients with SLE (groups1 and 2). There was no difference between all three studied cytocines or sFlt-1/PlGF ratio when the results were compared between inactive and active SLE. In this pilot study, pregnant patients with SLE and PE had lower mean serum levels of VEGF and PlGF and higher mean serum levels of sFlt-1 and sFlt-1/PlGF ratio than pregnant patients with inactive SLE and active SLE nephritis. The results suggest that SLE patients with PE have the same angiogenic and antiangiogenic profile of patients with PE without lupus. Further studies with more patients are needed to confirm the use of angiogenic and antiangiogenic factors as potential tools to differentiate PE from SLE nephritis in clinical practice. Download : Download high-res image (443KB) Download : Download full-size image

25 Low dose aspirin corrects in vitro the placental cell derived sFlt-1/PlGF imbalance characteristic of preeclampsia

Introduction Preeclampsia (PE) is a multi-system disorder typically involving pregnancy-induced maternal hypertension and proteinuria presenting in the second half of pregnancy. The placenta is the key organ responsible for the pathogenesis of PE, with altered release of placenta-derived anti- (eg sFlt-1) and pro- (eg PlGF) angiogenic proteins implicated in maternal PE pathophysiological events. Epidemiological data indicate that low-dose aspirin (LDA) commenced before 16 weeks gestation is clinically effective in preventing PE, but its mechanism of action is unclear. We hypothesized that the prophylactic efficacy of LDA treatment may be associated with a corrective effect of LDA on the altered release of placenta-derived angiogenic proteins seen in PE pregnancies. Objective To test this hypothesis, we examined the effects of pregnancy sera from normotensive and PE cases with and without LDA on the in vitro production and release of sFlt-1 and PlGF from a trophoblast-derived cell line (BeWo). Methods The effects of LDA (5 μg/mL) following treatment of BeWo cells in vitro with either PE or normotensive serum on the expression of angiogenic proteins sFlt-1 and PlGF were determined using real-time PCR. The secretion of these angiogenic proteins in the conditioned media was screened with a commercially designed protein array. Candidate protein validation was then performed using an ELISA. All experiments were performed in triplicate at least six independent occasions. A p-value Results Angiogenic profiling at the mRNA level identified significantly increased expression of Flt-1 [Normotensive: 1.04 ± 0.03 vs PE: 2.68 ± 0.07, p Conclusion Increased placental release of the anti-angiogenic protein sFlt-1 and reduced placental release of the pro-angiogenic protein PlGF into the maternal circulation are key contributors to the maternal vascular endothelial cell dysfunction that is central to the development of the maternal syndrome of PE. The ability of LDA to rebalance these key angiogenic proteins in an in vitro trophoblast cell-line model may offer an important mechanistic insight at a molecular level regarding the clinical utility of LDA in preventing PE.

High-circulating Tie2 Is Associated With Pathologic Complete Response to Chemotherapy and Antiangiogenic Therapy in Breast Cancer

Vascular endothelial growth factor (VEGF) is a central mediator of angiogenesis in breast cancer. Research in antiangiogenic cancer treatment has been marked by the development of the monoclonal antibody bevacizumab, which targets VEGF in many solid tumors. As patients do not equally benefit from bevacizumab, it has become necessary to define the profile of patients who will benefit from the drug. We have conducted a prospective phase II study in 39 patients using bevacizumab in breast cancer in the neoadjuvant setting, and found improved pathologic complete response (pCR) when bevacizumab was added to chemotherapy in patients with hormone receptor negative and invasive ductal carcinoma. Blood samples were collected at baseline and serially while patients were on treatment. Circulating angiogenesis-related proteins angiopoietin (ANG)1, ANG2, basic fibroblast growth factor, IL-1a, matrix metalloproteinase 9, platelet derived growth factor - BB, platelet endothelial cell adhesion molecule -1, Tie2, VEGF, and vascular endothelial growth factor receptor 2 were measured at baseline and during treatment. This correlative study was conducted to identify specific serum angiogenic factor profiles that might be associated with pCR in the neoadjuvant setting in breast cancer patients receiving bevacizumab and chemotherapy. Elevated baseline serum Tie2 and basic fibroblast growth factor were associated with pCR in response to this combination. Changes in serum levels of these proteins were seen during treatment but were not significantly different between the pCR and non-pCR groups. Baseline-circulating Tie2 levels may help distinguish patients who will have pCR from those who will not and may form the basis for future development of antiangiogenic therapy in breast cancer. Larger studies are needed to validate these findings. ClinicalTrials.gov Identifier: NCT00203502.

Inhibition of PAI-1 Limits Tumor Angiogenesis Regardless of Angiogenic Stimuli in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor that secretes various angiogenic factors. The main inhibitor of plasminogen activators, PAI-1 (SERPINE1), has been implicated in tumor progression and angiogenesis, and high PAI-1 expression has been associated with poor prognosis in MPM patients. In this study, we examined the antiangiogenic effects of PAI-1 inhibition in MPM. We administered the PAI-1 inhibitor, SK-216, to orthotopic mouse models in which MPM cells expressing high levels of VEGF (VEGFA) or bFGF (FGF2) were intrapleurally transplanted. SK-216 administration reduced tumor weights and the degree of angiogenesis in intrapleural tumors, irrespective of their angiogenic expression profiles. In addition, a combination of SK-216 and the chemotherapeutic agent cisplatin significantly reduced tumor weights compared with monotherapy, prolonging the survival of animals compared with cisplatin treatment alone. Furthermore, SK-216 inhibited migration and tube formation of cultured human umbilical vein endothelial cells induced by various angiogenic factors known to be secreted by MPM. These findings suggest that PAI-1 inactivation by SK-216 may represent a general strategy for inhibiting angiogenesis, including for the treatment of MPM. Cancer Res; 76(11); 3285-94. ©2016 AACR.

Investigation of GREMLIN 1, COL15A1 immunoreactivity and the relationship between microvessel density and GREMLIN 1 in papillary renal cell carcinoma and chromophobe renal cell carcinoma

Objective: The current study aimed to investigate expressions of Gremlin 1 (GREM1) (a bone morphogenetic protein antagonist and a proangiogenic factor) and COL15A1 (Collagen type XV alpha-1, encodes the proteoglycans located in various human tissues and particularly in the basement membrane) immunohistochemically in papillary renal cell carcinoma (PRCC) and chromophobe renal cell carcinoma (CRCC) in order to assess the relationship between these markers and said tumors and also explore associations of GREM1 with angiogenesis, tumor necrosis and tumor diameter by looking at the microvascular density (MVD) through the expression of COL15A1 in vascular endothelium. Material and Method: GREM1 and COL15A1 expressions were investigated in 20 PRCC and 39 CRCC patients. Cytoplasmic staining with GREM1 and COL15A1 was examined. Microvascular structures stained with COL15A1 were examined in order to evaluate angiogenic profile. Results: In CRCC, GREM1 staining was statistically significant in tumor tissues compared to intact tissues (p=0.006). The relationship between MVD and GREM1 staining was statistically significant in PRCC (p=0.007). Cytoplasmic staining with COL15A1 observed in PRCC was statistically significant (p=0.005). Conclusion: Positive GREM1 staining observed in both tumor groups and much higher expression of this marker particularly in the tubular epithelium of the neighboring normal tissue supports our argument that this gene might be a tumor suppressor gene.

Assessment of Plasma Cytokine Profile Changes in Bevacizumab-Treated Retinopathy of Prematurity Infants

We compared changes of plasma angiogenesis cytokine profiles in infants who were treated with intravitreal injection of bevacizumab (IVB) for type 1 retinopathy of prematurity (ROP) with age-matched preterm non-ROP infants. Thirteen infants with type 1 ROP and 13 age-matched preterm non-ROP infants were included. Blood samples were collected prior to treatment (time 0) and 6 weeks after the treatment (time 42). Plasma levels of nine cytokines from the angiogenesis growth factor panel and seven soluble cytokine receptors were measured using a magnetic multiplex assay. Plasma cytokine profiles changed from time 0 to time 42 in both groups. In bevacizumab-treated ROP infants, the following plasma angiogenesis growth factor and soluble cytokine receptor levels decreased significantly: soluble VEGF-A (sVEGF-A; P = 0.0001), sVEGF-D (P = 0.04), angiopoietin-2 (Ang-2; P = 0.002), sVEGF receptor 1 (R1) and R2 (P = 0.005), soluble IL-6 receptor (sIL-6R; P = 0.002), soluble glycoprotein 130 (spg130; P = 0.0001), and soluble TNF receptor (sTNFR) I and II (P = 0.0001). The following factors and receptors increased significantly: sVEGF-C (P = 0.05), placental growth factor (PlGF; P = 0.02), endothelin-1 (ET-1; P = 0.0001), and FGF-1 (P = 0.02). At time 42, sVEGF-A, sgp130, sIL-6R, sTNFR I, and sTNFR II were lower, and ET-1 level was higher, in bevacizumab-treated ROP infants compared to age-matched non-ROP infants. The results suggest that bevacizumab treatment resulted in significant angiogenic cytokine profile changes in infants with severe ROP. The long-term clinical impact of these changes should be studied carefully.

The Influence Of Obesity‐Associated Metabolic Disturbance On The Profile Of Angiogenic Regulators And Capillary Number Within Skeletal Muscle

Skeletal muscle capillaries are critical determinants of nutrient delivery and utilization and capillary network density is thought to correlate with insulin sensitivity. Conflicting data exist regarding the consequence of obesity‐associated metabolic disturbances on skeletal muscle capillarization, with positive, negative and no effect having been reported. The purpose of this investigation is to assess the influence of obesity induced by high fat feeding or by leptin receptor mutation, on the angiogenic profile within the skeletal muscle microenvironment, and ultimately, on muscle capillarization. In FVB/n mice fed a high‐fat (HF) or normal chow (NC) diet for 16‐weeks, mRNA levels of VEGF‐A did not change (NC: 1.06 vs. HF: 1.13; P>0.05) but VEGF protein levels were elevated (NC: 81 vs HF: 104 pg/mg; P =0.02). Relative FoxO1 protein levels increased in capillaries isolated from muscle of HF vs. NC mice (NC: 0.67 vs HF: 1.55; P =0.03). Capillary‐to‐fibre ratio (C:F) was not altered in HF‐ compared to NC‐ fed mice (2.22 and 2.04, P=0.34). In contrast, 13 week old mice with leptin receptor activity deficiency (Leprdb) had a significant reduction in VEGF‐A mRNA (1.85 vs. 1.09, P=0.03) and protein (WT: 98 vs Leprdb: 86 pg/mg; P<0.05) relative to age‐matched C57BL/6 (WT) mice. Both FoxO1 (0.58 vs. 0.86) and THBS1 (0.37 vs. 0.81) mRNA levels tended to increase in Leprdb vs. WT mice. Skeletal muscle C:F was substantially reduced in Leprdb vs. WT mice (1.92 vs. 1.39, P<0.001). Comprehensive profiling of angiogenic regulators will be conducted by microarray analysis. The skeletal muscle microenvironment differs substantially between HF and Leprdb models. Our data suggest that muscle capillarization is maintained during HF feeding due to a balance between pro and anti‐angiogenic factors, which may include VEGF‐A and FoxO1. This balance is disrupted in leptin receptor mutant mice, which results in a loss of capillaries and raises the intriguing possibility that leptin signaling itself modulates muscle capillarization. Thus, the Leprdb model may not be a suitable model to study obesity‐induced vascular alterations in skeletal muscle. Funded by CIHR.

Anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cytotrophoblast cell model

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis‐like factor, marinobufagenin (MBG) has been implicated as a causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. We have identified a novel human monoclonal antibody with higher specificity than Digibind for MBG. We assessed the attenuation of MBG‐induced CTBs dysfunction by three anti‐MBG antibodies: 206–208, H1L2, and 3e9.MethodsA panel of anti‐MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206–208, H1L2). H1L2 was a humanized version of previously described anti‐MBG murine antibody 3e9. 206–208 was identified in a human phage antibody library. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h. Some cells were pretreated with 206–208, H1L2, or 3e9 for 2h, while others were co‐treated with these antibodies prior to MBG treatment. Culture media were collected for analysis of pro‐angiogenic and anti‐angiogenic factors. Cell viability was measured using a CellTiter Assay kit. Cell lysates were utilized to evaluate the expression of proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms‐like tyrosine kinase‐1 (sFlt‐1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.ResultsMBG down‐regulated PCNA and up‐regulated p38 phosphorylation in CTBs treated with ≥1 nM MBG compared to basal (DMSO treatment) (*p < 0.05 for each). Secretion of sFlt‐1 and sEng were increased, while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG (*p < 0.05 for each). Both anti‐MBG antibodies pretreatment and co‐treatment significantly up‐regulated PCNA and down‐regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p < 0.05 for each). The anti‐proliferative and anti‐angiogenic effects of MBG were not due to a cytotoxic effect, as evaluated by a cell viability assay. MBG at 0.1 nM had no effect.ConclusionsWe found that all 3 anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cultured CTBs. Here we describe for the first time two fully human anti‐MBG antibodies, which can be targeted as therapeutic agents for the development of innovative treatment strategies for preE and potentially other disorders involving aberrant MBG signaling.Support or Funding InformationNIH Small Business Innovation Research (SBIR) : 1R43HD079238‐01

Serum angiogenic profile in abnormal placentation

Objective: To evaluate the circulating soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) levels in women with abnormal placentation and to compare the data with the results of women with normal pregnancy.Material and methods: Serum biomarkers of angiogenesis and maternal and perinatal characteristics of 68 pregnant women, all in the third trimester, who were diagnosed to have vaginal bleeding due to complete placenta previa with and without concomitant placenta accreta, increta and percreta as the study group and 30 pregnant women without any placentation abnormality who eventually delivered at ≥37 weeks of gestational age as the control group were evaluated.Results: There was no statistical difference in the maternal serum values of sFlt1, PlGF, sFlt1/PlGF ratio and VEGF in groups with placental abnormality as compared to controls. Not even a single case of preeclampsia and intrauterine fetal growth restriction was encountered in the study group.Conclusion: We demonstrated that regardless of the localization and the degree of the myometrial invasion of the placenta in the uterus, the circulatory biomarkers of angiogenesis and vascularization were comparable.

Abstract TP119: Angiogenic Factor Profiling in Medical Treatment of Intracranial Atherosclerosis (ICAS)

Objective: The purpose of this study was to identify circulating angiogenic factor profiles associated to success or failure of intensive medical treatment in ICAS patients. Methods: Prospective observational study of circulating angiogenic factors in 72 patients with ICAS treated medically. Twenty-one pro and antiangiogenic factors were isolated from plasma in patients with intracranial arterial stenosis greater than 70% due to ICAS. Patients were followed for 6 months and mRS were collected. Failure to medical management was defined as presence of TIA or stroke within 30 days of presentation. Levels of angiogenic factors were determined by plasma multiplex sandwich ELISA. Samples were run in duplicate with variation coefficient lower than 20%. Angiogenic profiles (APs) were defined using Principal Component Analysis (PCA). Seven profiles were selected (Eigenvalue greater than 1.0). A logistic regression model was built for the outcome variable medical management failure/success using the 7 APs as predictors. An ordinal logistic regression model was built to predict the outcome variable mRS at 6 months using age, gender, baseline mRS and APs as predictors. Results: Seventy-two patients with ICAS were included. Mean age was 61.8 (SD 12.3)yrs. There were 53% females. The predominant region of stenosis was the MCA. Of the 7 APs defined by PCA, the regression model identified AP-4 as significantly associated to failure of medical management (P=0.02). AP-4 was characterized by high levels of HGF and the anti-angiogenic factors VEGFR1, Endostatin, Angiostatin, VEGFR3, and TSP2. In the long-term follow up, AP-4 was positively associated with mRS, independently of baseline mRS and age (P=0.002). Conclusion: In this extensive prospective study of angiogenic factors in patients with ICAS, a profile characterized by elevation of HGF and several circulating anti-angiogenic factors was strongly associated with failure to medical management and poor outcomes at 6 months.

Abstract TMP15: Circulating Antiangiogenic Profile Predicts Lower Degrees of Neovascularization After Indirect Revascularization With Encephaloduroarteriosynangiosis (EDAS) in Patients With Intracranial Atherosclerosis (ICAS)

Objective: To evaluate the hypothesis that circulating pro and antiangiogenic profiles predict the degree of angiographic neovascularization in patients undergoing EDAS for the treatment of symptomatic ICAS. Methods: This is a prospective observational study of circulating angiogenic factors in patients with arterial stenosis greater than 70% due to ICAS. All patients received intensive medical management. Patients with persistent symptoms underwent in addition EDAS revascularization. Twenty-one angiogenic factors were analyzed in serum samples collected at baseline, before surgery, using multiplex ELISA. All samples were run in duplicate and accepted as valid if the intersample variability was less than 20%. Patients have angiographic follow up 6 months after EDAS and a Neovascularization Index (NI) based on Perren grades and ASITN collaterals were calculated. Angiogenic profiles (APs) were defined using Principal Component Analysis (PCA). Seven profiles were selected for each time-point (Eigenvalue greater than 1.0). A regression model was built for the outcome variable NI using as predictor variables age, smoking history diabetes, and the 7 APs. Forward stepwise modeling with minimum BIC stopping rule was used to select the best fitted model. Results: Fifteen patients were enrolled. Mean age was 62.1 ± 12.3, 75% were females. The regression model demonstrated significant associations of age, diabetes and the AP 1-6 with the NI. Whole model P=0.023, Rsquare=0.93. The solution of the regression formula for the significant AP components revealed a strong negative association of the antiangiogenic factors endostatin and angiostatin (regression parameters -1.8 and -4.5, respectively, p=0.02). At 6 months follow up no patients had suffer strokes. Conclusion: In this prospective study of angiogenic factors in patients with ICAS undergoing EDAS revascularization, we have found that baseline levels of the antiangiogenic factors endostatin and angiostatin are significantly negatively associated to the degree of postoperative revascularization at 6 months. This finding supports the concept that neutralization of antiangiogenic factors may serve as a new pharmacological target to treat cerebral ischemia in patients with ICAS.

Multipotent adult progenitor cells on an allograft scaffold facilitate the bone repair process

Multipotent adult progenitor cells are a recently described population of stem cells derived from the bone marrow stroma. Research has demonstrated the potential of multipotent adult progenitor cells for treating ischemic injury and cardiovascular repair; however, understanding of multipotent adult progenitor cells in orthopedic applications remains limited. In this study, we evaluate the osteogenic and angiogenic capacity of multipotent adult progenitor cells, both in vitro and loaded onto demineralized bone matrix in vivo, with comparison to mesenchymal stem cells, as the current standard. When compared to mesenchymal stem cells, multipotent adult progenitor cells exhibited a more robust angiogenic protein release profile in vitro and developed more extensive vasculature within 2 weeks in vivo. The establishment of this vascular network is critical to the ossification process, as it allows nutrient exchange and provides an influx of osteoprogenitor cells to the wound site. In vitro assays confirmed the multipotency of multipotent adult progenitor cells along mesodermal lineages and demonstrated the enhanced expression of alkaline phosphatase and production of calcium-containing mineral deposits by multipotent adult progenitor cells, necessary precursors for osteogenesis. In combination with a demineralized bone matrix scaffold, multipotent adult progenitor cells demonstrated enhanced revascularization and new bone formation in vivo in an orthotopic defect model when compared to mesenchymal stem cells on demineralized bone matrix or demineralized bone matrix–only control groups. The potent combination of angiogenic and osteogenic properties provided by multipotent adult progenitor cells appears to create a synergistic amplification of the bone healing process. Our results indicate that multipotent adult progenitor cells have the potential to better promote tissue regeneration and healing and to be a functional cell source for use in orthopedic applications.

Differential Expression of Angiogenic Gene Networks during Post-natal Lung Alveolarization

Prematurity of birth is the leading cause of death in children under 5 years of age. Preterm birth rates are steadily increasing across the globe due to advancements in medical knowledge and procedures that allow progressively earlier fetuses to survive outside of the mother’s womb. Bronchopulmonary dysplasia is a chronic lung disorder that can result from mechanical ventilation and long-term oxygen use in preterm neonates. This condition is characterized by disrupted pulmonary alveolar septation, alveolar hypoplasia, and fewer, larger alveoli that lead to decreased surface area available for gas exchange. Despite notable advances in preterm care, bronchopulmonary dysplasia results in a large number of short- and long-term morbidities, thus more advanced treatments that facilitate lung development in this subset of pediatric patients are sorely needed. In this study, we examined alterations in the gene expression profiles of 81 genes involved in angiogenesis during the process of normal alveolarization in mouse lungs. Our data revealed that alveolarization can be largely divided into early (1 week postnatal) and late (4 to 8 weeks postnatal) stages based on angiogenic gene expression profiles. In summary, our findings provide molecularlevel data illustrating the dynamic alterations in pulmonary angiogenesis during postnatal alveolarization. This knowledge can be used to provide a better understanding of normal lung development, and set the stage for discovering targeted therapies that can assist in pulmonary functioning for preterm infants.

Safety and pharmacokinetics of pravastatin used for the prevention of preeclampsia in high-risk pregnant women: a pilot randomized controlled trial

Preeclampsia complicates approximately 3-5% of pregnancies and remains a major cause of maternal and neonatal morbidity and mortality. It shares pathogenic similarities with adult cardiovascular disease as well as many risk factors. Pravastatin, a hydrophilic, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, has been shown in preclinical studies to reverse various pathophysiological pathways associated with preeclampsia, providing biological plausibility for its use for preeclampsia prevention. However, human trials are lacking. As an initial step in evaluating the utility of pravastatin in preventing preeclampsia and after consultation with the US Food and Drug Administration, we undertook a pilot randomized controlled trial with the objective to determine pravastatin safety and pharmacokinetic parameters when used in pregnant women at high risk of preeclampsia. We conducted a pilot, multicenter, double-blind, placebo-controlled, randomized trial of women with singleton, nonanomalous pregnancies at high risk for preeclampsia. Women between 12(0/7) and 16(6/7) weeks' gestation were assigned to daily pravastatin 10 mg or placebo orally until delivery. Primary outcomes were maternal-fetal safety and pharmacokinetic parameters of pravastatin during pregnancy. Secondary outcomes included rates of preeclampsia and preterm delivery, gestational age at delivery, birthweight, and maternal and cord blood lipid profile (clinicaltrials.gov identifier NCT01717586). Ten women assigned to pravastatin and 10 to placebo completed the trial. There were no differences between the 2 groups in rates of study drug side effects, congenital anomalies, or other adverse or serious adverse events. There was no maternal, fetal, or neonatal death. Pravastatin renal clearance was significantly higher in pregnancy compared with postpartum. Four subjects in the placebo group developed preeclampsia compared with none in the pravastatin group. Although pravastatin reduced maternal cholesterol concentrations, umbilical cord cholesterol concentrations and infant birthweight were not different between the groups. The majority of umbilical cord and maternal pravastatin plasma concentrations at the time of delivery were below the lower limit of quantification of the assay. Pravastatin use was associated with a more favorable pregnancy angiogenic profile. This study provides preliminary safety and pharmacokinetic data regarding the use of pravastatin for preventing preeclampsia in high-risk pregnant women. Although the data are preliminary, no identifiable safety risks were associated with pravastatin use in this cohort. This favorable risk-benefit analysis justifies using pravastatin in a larger clinical trial with dose escalation.

The Effect of Donor-Dependent Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells following Focal Cerebral Ischemia in Rats

Stroke is an ischemic disease caused by clotted vessel-induced cell damage. It is characterized by high morbidity and mortality and is typically treated with a tissue plasminogen activator (tPA). However, this therapy is limited by temporal constraints. Recently, several studies have focused on cell therapy as an alternative treatment. Most researches have used fixed donor cell administration, and hence, the effect of donor-dependent cell administration is unknown. In this study, we administered 3 types of donor-derived human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in the ischemic boundary zone of the ischemic stroke rat model. We then performed functional and pathological characterization using rotarod, the limb placement test, and immunofluorescent staining. We observed a significant decrease in neuron number, and notable stroke-like motor dysfunction, as assessed by the rotarod test (~40% decrease in time) and the limb placement test (4.5 point increase) in control rats with ischemic stroke. The neurobehavioral performance of the rats with ischemic stroke that were treated with hUCB-MSCs was significantly better than that of rats in the vehicle-injected control group. Regardless of which donor cells were used, hUCB-MSC transplantation resulted in an accumulation of neuronal progenitor cells, and angiogenic and tissue repair factors in the ischemic boundary zone. The neurogenic and angiogenic profiles of the 3 types of hUCB-MSCs were very similar. Our results suggest that intraparenchymal administration of hUCB-MSCs results in significant therapeutic effects in the ischemic brain regardless of the type of donor.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Enhanced Cardiac Regenerative Ability of Stem Cells After Ischemia-Reperfusion Injury: Role of Human CD34+ Cells Deficient in MicroRNA-377

BackgroundMicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress. ObjectivesThe aim of this study was to explore the role of miR in human CD34+ cell (hCD34+) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions. MethodsIn response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction–based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34+ cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice. ResultsThe miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34+ cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control–transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34+ cells in immunodeficient mice, promoting neovascularization (at 28 days, post–I-R) and lower interstitial fibrosis, leading to improved left ventricular function. ConclusionsThese findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34+ cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis.

Scaling up the ex vivo expansion of human circulating CD34+progenitor cells with upregulation of angiogenic and anti-inflammatory potential

Background aimsThe therapeutic application of CD34+ circulating progenitor cells (which includes endothelial progenitor cells) has been hampered by the quantity and quality of isolated circulating CD34+ cells from the patient's peripheral blood. Our group had previously established a suspension culture system for human CD34+ cells, with increased quantity and quality (QQ) of the angiogenic cell product. We successfully scaled up the expansion process with the use of culture bags because there is the need to move toward a dynamic and fully controlled bioreactor system to meet Good Manufacturing Practice (GMP) standards and attain clinically meaningful cell doses in a time- and cost-effective way. MethodsCD34+ cells isolated from mobilized peripheral blood of healthy donors were expanded ex vivo for 7 days in QQ medium (serum-free) in cell culture bags (30 mL) and pre- and post-expansion cells were characterized by means of flow cytometry and quantitative polymerase chain reaction; angiogenic potential was assessed by use of the in vitro tube formation assay. ResultsOur data show effective expansion of the cultured population (7-fold) while maintaining the stem/progenitor content and increasing the endothelial population. Moreover, post-expanded cells showed higher tube formation capacity compared with pre-expanded cells. In addition, an upregulation of the anti-inflammatory gene expression and a downregulation of pro-inflammatory genes were observed, which suggests that the increase in angiogenic potential is not paired with an increase in the inflammatory profile. ConclusionsThe QQ expansion method was successfully scaled up to cell culture bags and was able to meet GMP standards, with a higher in vitro angiogenic profile.

Urinary tract infection during pregnancy, angiogenic factor profiles, and risk of preeclampsia

Despite decades of research, and much progress in discernment of biomarkers in the maternal circulation, the pathogenesis of preeclampsia (PE) remains elusive. The pathophysiology of PE is believed to involve aberrant placentation and an associated increase in systemic inflammation. In this conceptualization, PE becomes more likely when the level of systemic inflammatory burden inherent in pregnancy itself exceeds the maternal capacity to compensate for this additional stress. If this is the case, then it is possible to hypothesize that conditions, such as infectious disease, that increase systemic inflammatory burden should also increase the risk of PE. As urinary tract infection (UTI) represents a common source of inflammation during pregnancy, we tested whether presence of UTI during pregnancy increased the odds of developing PE. Prior work has documented this association. However many of these studies were limited by small cohort sizes and insufficient control for covariates. The present study is a secondary analysis of a robust contemporary obstetrical cohort recruited to examine the ability of longitudinally sampled maternal angiogenic concentrations to predict PE. We hypothesize that the occurrence of UTI during a pregnancy is associated with the later occurrence of PE in that pregnancy. As PE is believed to be associated with aberrations in systemic angiogenic levels (placental growth factor and soluble isoform of VEGF receptor), we further hypothesize that there will be significant interactions between maternal angiogenic protein levels and the occurrence of UTI. Women aged ≥18 years (n = 2607) were recruited and followed up prospectively from the initiation of prenatal care through delivery at 3 regional academic centers. PE was defined by American Congress of Obstetricians and Gynecologists criteria and was independently validated by a panel of physicians. UTI was defined by the presence of clinical symptoms necessitating treatment in addition to supportive laboratory evidence. Multivariate logistic regression models were used and controlled for maternal age, race, parity, body mass index, hypertension, diabetes, in vitro fertilization, and smoking status. There were 129 women with diagnosed UTIs and 235 with PE. Patients with UTI in pregnancy had higher rates of PE (31.1% vs 7.8%, P < .001) compared to those without reported UTI. The mean gestational age (SD) for UTI diagnosis in PE cases and controls was 25.6 (10.4) and 21.9 (10.9) weeks, respectively (P = .08). The unadjusted odds ratio for PE in the setting of UTI was 5.29 (95% confidence interval, 3.54-7.89). After controlling for confounders, UTI was associated with an odds ratio for PE of 3.2 (95% confidence interval, 2.0-5.1). Presence of UTI in pregnancy, particularly in the third trimester, is strongly associated with PE. This association supports the hypothesis that the risk of PE is enhanced by an increased maternal inflammatory burden. Prophylaxis against UTI represents a potentially low-cost global intervention to slow or halt the development of PE.