Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Decreased vascular reactivity associated with increased IL-8 in 6-month-old infants of mothers with pre-eclampsia.

BACKGROUND: Offspring born to mothers with pre-eclampsia (Pre-E) suffer higher risks of adult cardiovascular diseases, suggesting that exposure to an antiangiogenic environment in-utero has a lasting impact on the development of endothelial function. The goal of this study is to test the hypothesis that in-utero exposure to Pre-E results in alterations of angiogenic factors/cytokines that negatively impact vascular development during infancy. METHODS: Infants born from mothers with and without Pre-E were recruited and followed up at 6 months. Plasma cytokines, blood pressure, microvessel density, and vascular reactivity were assessed. RESULTS: 6-month-old infants born to mothers with Pre-E had unchanged blood pressure (p = 0.86) and microvessel density (p = 0.57). Vascular reactivity was decreased in infants born to mothers with Pre-E compared to infants born to healthy mothers (p = 0.0345). Interleukin 8 (IL-8) (p = 0.03) and Angiopoeitin-2 (Ang-2) (p = 0.04) were increased in infants born to mothers with Pre-E. We observed that higher IL-8 was associated with lower vascular reactivity (rho = -0.14, p < 0.0001). CONCLUSION: At 6 months of age, infants born to mothers with Pre-E had impaired vascular reactivity and higher IL-8 and Ang-2, but similar blood pressure and microvessel density compared to infants born to non-Pre-E mothers. IMPACT STATEMENT: Changes in cord blood antiangiogenic factors are documented in infants of mothers with pre-eclampsia and may contribute to offspring risks of adult cardiovascular disease. How these factors evolve during early infancy and their correlation with offspring vascular development have not been studied. This study found that 6-month-old infants born to mothers with pre-eclampsia had decreased vascular reactivity, which was correlated with higher IL-8. These findings underscore the lasting impact of maternal pre-eclampsia on offspring vascular development and highlight the need for long-term follow-up in children born to mothers with pre-eclampsia.

The Effects of Preeclamptic Milieu on Cord Blood Derived Endothelial Colony-Forming Cells.

Preeclampsia is one of the leading causes of infant and maternal mortality worldwide. Many infants born from preeclamptic pregnancies are born prematurely with higher risk of developing cardiovascular later in their life. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). To gain insight into this, cord blood derived ECFCs isolated from preeclamptic pregnancies (PRECs) were analyzed and compared to their healthy counterparts. While PRECs preserve key endothelial markers, they upregulate several markers associated with oxidative stress and inflammatory response. Compared to ECFCs, PRECs also exhibit lower migratory behaviors and impaired angiogenic potential. Interestingly, treatment of neuropilin-1 can improve tube formation in vitro. Collectively, this study reports that preeclamptic milieu influence phenotypes and functionality of PRECs, which can be rejuvenated using exogenous molecules. Promising results from this study warrant future investigations on the prospect of the rejuvenated PRECs to improve lung function of infants born from preeclamptic pregnancies.

Long-term childhood outcomes for babies born at term who were exposed to antenatal corticosteroids.

BACKGROUND: Antenatal corticosteroids improve neonatal outcomes when administered to infants who are at risk of preterm delivery. Many women who receive antenatal corticosteroids for threatened preterm labor proceed to deliver at term. Thus, long-term outcomes should be evaluated for term-born infants who were exposed to antenatal corticosteroids in utero. OBJECTIVE: This study aimed to compare long-term outcomes between term-born children aged ≥5 years who were born to women who received antenatal corticosteroids for threatened preterm labor and children whose mothers were also evaluated for threatened preterm labor but did not receive antenatal corticosteroids. STUDY DESIGN: We performed a retrospective cohort study of children born at ≥37 weeks' gestation, aged ≥5 years, and born to mothers diagnosed with threatened preterm labor during pregnancy. The primary exposure of interest was receiving antenatal corticosteroids. Among the collected childhood medical conditions, the primary outcome of interest was a diagnosis of asthma. RESULTS: Of the 3556 term-born children aged ≥5 years, 629 (17.6%) were exposed to antenatal corticosteroids (all betamethasone), and 2927 (82.3%) were controls whose mothers were evaluated for threatened preterm birth but did not get antenatal corticosteroid injections. Women receiving antenatal corticosteroids had higher rates of maternal comorbidities (diabetes mellitus, hypertension; P≤.01). Antenatal corticosteroid-exposed children had no difference in diagnosis of asthma (12.6% vs 11.6%), attention deficit disorder, or developmental delay (P=.47, .54, and .10, respectively). Controlling for maternal and neonatal characteristics, asthma was not different between those exposed to antenatal corticosteroids and controls (odds ratio, 1.05; 95% confidence interval, 0.79-1.39). The odds of the child's weight percentile being <10% were increased for antenatal corticosteroid-exposed children born at term (odds ratio, 2.00; 95% confidence interval, 1.22-3.25). CONCLUSION: Children born at term who were exposed to antenatal corticosteroids may have increased odds of being in a lower growth percentile than those not exposed. However, rates of diagnoses such as asthma, developmental delay, and attention deficit disorders were not different.

Forced expiratory flows and diffusion capacity in infants born from mothers with pre-eclampsia.

RATIONALE: Animal models suggest pre-eclampsia (Pre-E) affects alveolar development, but data from humans are lacking. OBJECTIVE: Assess the impact of Pre-E on airway function, diffusion capacity, and respiratory morbidity in preterm and term infants born from mothers with Pre-E. METHODS: Infants born from mothers with and without Pre-E were recruited for this study; term and preterm infants were included in both cohorts. Respiratory morbidity in the first 12 months of life was assessed through monthly phone surveys. Raised volume rapid thoracoabdominal compression and measurement of diffusion capacity of the lung to carbon monoxide (DLCO) were performed at 6 months corrected age. MEASUREMENTS AND MAIN RESULTS: There were 146 infants in the Pre-E cohort and 143 in the control cohort. The Pre-E cohort was further divided into nonsevere (N = 41) and severe (N = 105) groups. There was no significant difference in DLCO and DLCO/alveolar volume among the three groups. Forced vital capacity was similar among the three groups, but the nonsevere Pre-E group had significantly higher forced expiratory flows than the other two groups. After adjusting for multiple covariates including prematurity, the severe Pre-E group had a lower risk for wheezing in the first year of life compared to the other two groups. CONCLUSIONS: Pre-E is not associated with reduced DLCO, lower forced expiratory flows, or increased wheezing in the first year of life. These results differ from animal models and highlight the complex relationships between Pre-E and lung function and respiratory morbidity in human infants.

Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells.

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including type-2 diabetes mellitus, hypertension, and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial function, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and unintended immune response of gene therapies. Here, we report a strategy to rejuvenate circulating vascular progenitor cells by conjugation of drug-loaded liposomal nanoparticles directly to the surface of GDM-exposed ECFCs (GDM-ECFCs). Bioactive nanoparticles can be robustly conjugated to the surface of ECFCs without altering cell viability and key progenitor phenotypes. Moreover, controlled delivery of therapeutic drugs to GDM-ECFCs is able to normalize transgelin (TAGLN) expression and improve cell migration, which is a critical key step in establishing functional vascular networks. More importantly, sustained pseudo-autocrine stimulation with bioactive nanoparticles is able to improve in vitro and in vivo vasculogenesis of GDM-ECFCs. Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potential. Promising results from this study warrant future investigations on the prospect of the proposed strategy to improve dysfunctional vascular progenitor cells in the context of other chronic diseases, which has broad implications for addressing various cardiovascular complications, as well as advancing tissue repair and regenerative medicine.

Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.

Are newborn outcomes different for term babies who were exposed to antenatal corticosteroids?

BACKGROUND: Antenatal corticosteroids improve newborn outcomes for preterm infants. However, predicting which women presenting for threatened preterm labor will have preterm infants is inaccurate, and many women receive antenatal corticosteroids but then go on to deliver at term. OBJECTIVE: This study aimed to compare the short-term outcomes of infants born at term to women who received betamethasone for threatened preterm labor with infants who were not exposed to betamethasone in utero. STUDY DESIGN: We performed a retrospective cohort study of infants born at or after 37 weeks' gestational age to mothers diagnosed as having threatened preterm labor during pregnancy. The primary neonatal outcomes of interest included transient tachypnea of the newborn, neonatal intensive care unit admission, and small for gestational age and were evaluated for their association with betamethasone exposure while adjusting for covariates using multiple logistic regression. RESULTS: Of 5330 women, 1459 women (27.5%) received betamethasone at a mean gestational age of 32.2±3.3 weeks. The mean age of women was 27±5.9 years and the mean gestational age at delivery was 38.9±1.1 weeks. Women receiving betamethasone had higher rates of maternal comorbidities (P<.001 for diabetes mellitus, asthma, and hypertensive disorder) and were more likely to self-identify as White (P=.022). Betamethasone-exposed neonates had increased rates of transient tachypnea of the newborn, neonatal intensive care unit admission, small for gestational age, hyperbilirubinemia, and hypoglycemia (all, P<.05). Controlling for maternal characteristics and gestational age at delivery, betamethasone exposure was not associated with a diagnosis of transient tachypnea of the newborn (adjusted odds ratio, 1.10; 95% confidence interval, 0.80-1.51), although it was associated with more neonatal intensive care unit admissions (adjusted odds ratio, 1.49; 95% confidence interval, 1.19-1.86) and higher odds of the baby being small for gestational age (adjusted odds ratio, 1.78; 95% confidence interval, 1.48-2.14). CONCLUSION: Compared with women evaluated for preterm labor who did not receive betamethasone, women receiving betamethasone had infants with higher rates of neonatal intensive care unit admission and small for gestational age. Although the benefits of betamethasone to infants born preterm are clear, there may be negative impacts for infants delivered at term.

Stereoselective Analysis of Methadone and EDDP in Laboring Women and Neonates in Plasma and Dried Blood Spots and Association with Neonatal Abstinence Syndrome.

OBJECTIVE: This pilot study evaluated the relationship between maternal and neonatal R- and S-methadone and R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) exposure and the severity of neonatal abstinence syndrome (NAS). The use of dried blood spots (DBS) as an alternative for plasma in assessing methadone and EDDP was also assessed. STUDY DESIGN: Women receiving methadone for medication assisted treatment of opioid use disorder during pregnancy were eligible for recruitment. Plasma and DBS samples were collected from mothers during labor, from cord blood, and from newborns during genetic screen. R-/S-methadone and EDDP were measured by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). Associations between methadone exposure, neonatal morphine requirements, and severity of NAS were examined. RESULTS: Twenty women and infants completed the study. Maternal methadone dose at delivery was 112 mg/day (range = 60-180 mg/day). Sixteen neonates experienced NAS requiring morphine; three also required phenobarbital. Higher cord blood concentrations of R-methadone, R- and S-EDDP were associated with higher maximum doses of morphine (p < 0.05). CONCLUSION: Maternal methadone and cord blood concentration at delivery are variable and may be potential markers of neonatal abstinence syndrome.

Hyperglycemia cooperates with Tet2 heterozygosity to induce leukemia driven by proinflammatory cytokine-induced lncRNA Morrbid.

Diabetes mellitus (DM) is a risk factor for cancer. The role of DM-induced hyperglycemic (HG) stress in blood cancer is poorly understood. Epidemiologic studies show that individuals with DM are more likely to have a higher rate of mutations in genes found in pre-leukemic hematopoietic stem and progenitor cells (pre-LHSPCs) including TET2. TET2-mutant pre-LHSPCs require additional hits to evolve into full-blown leukemia and/or an aggressive myeloproliferative neoplasm (MPN). Intrinsic mutations have been shown to cooperate with Tet2 to promote leukemic transformation. However, the extrinsic factors are poorly understood. Using a mouse model carrying Tet2 haploinsufficiency to mimic the human pre-LHSPC condition and HG stress, in the form of an Ins2Akita/+ mutation, which induces hyperglycemia and type 1 DM, we show that the compound mutant mice developed a lethal form of MPN and/or acute myeloid leukemia (AML). RNA-Seq revealed that this was due in part to upregulation of proinflammatory pathways, thereby generating a feed-forward loop, including expression of the antiapoptotic, long noncoding RNA (lncRNA) Morrbid. Loss of Morrbid in the compound mutants rescued the lethality and mitigated MPN/AML. We describe a mouse model for age-dependent MPN/AML and suggest that hyperglycemia acts as an environmental driver for myeloid neoplasms, which could be prevented by reducing expression levels of the inflammation-related lncRNA Morrbid.

Osteocyte Vegf-a contributes to myeloma-associated angiogenesis and is regulated by Fgf23.

Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.

Effects of Pregnancy on the Pharmacokinetics of Metformin.

This study's primary objective was to fully characterize the pharmacokinetics of metformin in pregnant women with gestational diabetes mellitus (GDM) versus nonpregnant controls. Steady-state oral metformin pharmacokinetics in pregnant women with GDM receiving either metformin monotherapy (n = 24) or a combination with glyburide (n = 30) as well as in nonpregnant women with type 2 diabetes mellitus (T2DM) (n = 24) were determined utilizing noncompartmental techniques. Maternal and umbilical cord blood samples were collected at delivery from 38 women. With both 500- and 1000-mg doses, metformin bioavailability, volume of distribution beta (V ß ), clearance, and renal clearance were significantly increased during pregnancy. In addition, in the women receiving metformin 500 mg, significantly higher metformin apparent oral clearance (CL/F) (27%), weight-adjusted renal secretion clearance (64%), and apparent oral volume of distribution beta (V ß /F) (33%) were seen during pregnancy. Creatinine clearance was significantly higher during pregnancy. Increasing metformin dose from 500 to 1000 mg orally twice daily significantly increased V ß /F by 28%, weight-adjusted V ß /F by 32% and CL/F by 25%, and weight-adjusted CL/F by 28% during pregnancy. Mean metformin umbilical cord arterial-to-venous plasma concentration ratio was 1.0 ± 0.1, venous umbilical cord-to-maternal concentration ratio was 1.4 ± 0.5, and arterial umbilical cord-to-maternal concentration ratio was 1.5 ± 0.5. Systemic exposure after a 500-mg dose of metformin was lower during pregnancy compared with the nonpregnant women with T2DM. However, in patients receiving metformin 1000 mg, changes in estimated bioavailability during pregnancy offset the changes in clearance leading to no significant change in CL/F with the higher dose. SIGNIFICANCE STATEMENT: Gestational diabetes mellitus complicates 5%-13% of pregnancies and is often treated with metformin. Pregnant women undergo physiological changes that alter drug disposition. Preliminary data suggest that pregnancy lowers metformin concentrations, potentially affecting efficacy and safety. This study definitively describes pregnancy's effects on metformin pharmacokinetics and expands the mechanistic understanding of pharmacokinetic changes across the dosage range. Here we report the nonlinearity of metformin pharmacokinetics and the increase in bioavailability, clearance, renal clearance, and volume of distribution during pregnancy.

Pharmacodynamics of Glyburide, Metformin, and Glyburide/Metformin Combination Therapy in the Treatment of Gestational Diabetes Mellitus.

In gestational diabetes mellitus (GDM), women are unable to compensate for the increased insulin resistance during pregnancy. Data are limited regarding the pharmacodynamic effects of metformin and glyburide during pregnancy. This study characterized insulin sensitivity (SI), ß-cell responsivity, and disposition index (DI) in women with GDM utilizing a mixed-meal tolerance test (MMTT) before and during treatment with glyburide monotherapy (GLY, n = 38), metformin monotherapy (MET, n = 34), or GLY and MET combination therapy (COMBO; n = 36). GLY significantly decreased dynamic ß-cell responsivity (31%). MET and COMBO significantly increased SI (121% and 83%, respectively). Whereas GLY, MET, and COMBO improved DI, metformin (MET and COMBO) demonstrated a larger increase in DI (P = 0.05) and a larger decrease in MMTT peak glucose concentrations (P = 0.03) than subjects taking only GLY. Maximizing SI with MET followed by increasing ß-cell responsivity with GLY or supplementing with insulin might be a more optimal strategy for GDM management than monotherapy.

Pharmacodynamics of Metformin in Pregnant Women With Gestational Diabetes Mellitus and Nonpregnant Women With Type 2 Diabetes Mellitus.

Gestational diabetes mellitus is a condition similar to type 2 diabetes mellitus (T2DM) in that patients are unable to compensate for the degree of insulin resistance, and both conditions are often treated with metformin. The comparative pharmacodynamic response to metformin in these 2 populations has not been studied. This study characterized insulin sensitivity, ß-cell responsivity, and disposition index following a mixed-meal tolerance test utilizing a minimal model of glucose, insulin, and C-peptide kinetics before and during treatment with metformin. The study included women with gestational diabetes mellitus (n = 34), T2DM (n = 14), and healthy pregnant women (n = 30). Before treatment, the gestational diabetes mellitus group had significantly higher baseline (45%), dynamic (68%), static (71%), and total ß-cell responsivity (71%) than the T2DM group. Metformin significantly increased insulin sensitivity (51%) as well as disposition index (97%) and decreased mixed-meal tolerance test peak glucose concentrations (8%) in women with gestational diabetes mellitus after adjustment for gestational age-dependent effects; however, in women with T2DM metformin only significantly affected peak glucose concentrations (22%) and had no significant effect on any other parameters. Metformin had a greater effect on the change in disposition index (Δ disposition index) in women with gestational diabetes mellitus than in those with T2DM (P = .01). In conclusion, response to metformin in women with gestational diabetes mellitus is significantly different from that in women with T2DM, which is likely related to the differences in disease severity.

Endothelial Colony-Forming Cell Function Is Reduced During HIV Infection.

BACKGROUND: Human immunodeficiency virus (HIV) may be related to cardiovascular disease through monocyte activation-associated endothelial dysfunction. METHODS: Blood samples from 15 HIV-negative participants (the uninfected group), 8 HIV-positive participants who were not receiving antiretroviral therapy (ART) (the infected, untreated group), and 15 HIV-positive participants who were receiving ART (the infected, treated group) underwent flow cytometry of endothelial colony-forming cells (ECFCs) and monocyte proportions. IncuCyte live cell imaging of 8 capillary proliferative capacity parameters were obtained from cord blood ECFCs treated with participant plasma. RESULTS: The ECFC percentage determined by flow cytometry was not different between the study groups; however, values of the majority of capillary proliferative capacity parameters (ie, cell area, network length, network branch points, number of networks, and average tube width uniformity) were significantly lower in infected, untreated participants as compared to values for uninfected participants or infected, treated participants (P < .00625 for all comparisons). CD14+CD16+ intermediate monocytes and soluble CD163 were significantly and negatively correlated with several plasma-treated, cord blood ECFC proliferative capacity parameters in the combined HIV-positive groups but not in the uninfected group. CONCLUSIONS: Cord blood ECFC proliferative capacity was significantly impaired by plasma from infected, untreated patients, compared with plasma from uninfected participants and from infected, treated participants. Several ECFC functional parameters were adversely associated with monocyte activation in the HIV-positive groups, thereby suggesting a mechanism by which HIV-related inflammation may impair vascular reparative potential and consequently increase the risk of cardiovascular disease during HIV infection.

Transgelin induces dysfunction of fetal endothelial colony-forming cells from gestational diabetic pregnancies.

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through the functional impairment of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). Previously, we showed that fetal ECFCs exposed to GDM have decreased vasculogenic potential and altered gene expression. In this study, we evaluate whether transgelin (TAGLN), which is increased in GDM-exposed ECFCs, contributes to vasculogenic dysfunction. TAGLN is an actin-binding protein involved in the regulation of cytoskeletal rearrangement. We hypothesized that increased TAGLN expression in GDM-exposed fetal ECFCs decreases network formation by impairing cytoskeletal rearrangement resulting in reduced cell migration. To determine if TAGLN is required and/or sufficient to impair ECFC network formation, TAGLN was reduced and overexpressed in ECFCs from GDM and uncomplicated pregnancies, respectively. Decreasing TAGLN expression in GDM-exposed ECFCs improved network formation and stability as well as increased migration. In contrast, overexpressing TAGLN in ECFCs from uncomplicated pregnancies decreased network formation, network stability, migration, and alignment to laminar flow. Overall, these data suggest that increased TAGLN likely contributes to the vasculogenic dysfunction observed in GDM-exposed ECFCs, as it impairs ECFC migration, cell alignment, and network formation. Identifying the molecular mechanisms underlying fetal ECFC dysfunction following GDM exposure is key to ascertain mechanistically the basis for cardiovascular disease predisposition later in life.

Mycophenolic acid induces senescence of vascular precursor cells.

OBJECTIVE: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. METHODS: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. RESULTS: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated ß-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. CONCLUSION: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Impact of Pregnancy History and 17-Hydroxyprogesterone Caproate on Cervical Cytokines and Matrix Metalloproteinases.

OBJECTIVE: The objective of this study was to evaluate the impact of pregnancy history and 17-hydroxyprogesterone caproate (17-OHPC) treatment on cervical fluid cytokines and matrix metalloproteinases (MMPs). STUDY DESIGN: Cervical fluid was obtained between 160/7 and 246/7 weeks from women with only prior term births (controls, n = 26), women with one or more prior spontaneous preterm births (SPTBs) choosing to receive 17-OHPC (17-OHPC, n = 24), or to not receive 17-OHPC (refusers, n = 12). Cervical fluid collections were repeated 2, 4, and 8 weeks after the first sample and concentrations of MMPs and cytokines were measured by multiplex immune assay. RESULTS: Among women whose earliest prior delivery occurred between 16 and 23 weeks, cervical fluid concentration of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α at baseline were significantly elevated when compared with cervical cytokines of women whose earliest delivery occurred between 32 and 36 weeks (relative risk ratio was 3.37 for IL-6 [95% confidence interval, CI, 1.08-10.53, p < 0.05], 2.81 for IL-10 [95% CI, 1.39-5.70, p < 0.05], and 6.34 for TNF-α [95% CI, 2.19-18.68, p < 0.001]). Treatment with 17-OHPC had no significant impact on these cytokines. CONCLUSION: The cervical fluid of women with a history of an early prior SPTB is characterized by inflammation that is unaffected by 17-OHPC.

Kinetic analyses of vasculogenesis inform mechanistic studies.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.