Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments.

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 µm-1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 µm-1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Cyclic nucleotide-dependent relaxation in human umbilical vessels.

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the ß-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (< 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels.

Umbilical cannulation optimizes circuit flows in premature lambs supported by the EXTra-uterine Environment for Neonatal Development (EXTEND).

KEY POINTS: Bronchopulmonary dysplasia is a disease of extreme prematurity that occurs when the immature lung is exposed to gas ventilation. We designed a novel 'artificial womb' system for supporting extreme premature lambs (called EXTEND) that obviates gas ventilation by providing oxygen via a pumpless arteriovenous circuit with the lamb submerged in sterile artificial amniotic fluid. In the present study, we compare different arteriovenous cannulation strategies on EXTEND, including carotid artery/jugular vein (CA/JV), carotid artery/umbilical vein (CA/UV) and umbilical artery/umbilical vein (UA/UV). Compared to CA/JV and CA/UV cannulation, UA/UV cannulation provided significantly higher, physiological blood flows to the oxygenator, minimized flow interruptions and supported significantly longer circuit runs (up to 4 weeks). Physiological circuit blood flow in UA/UV lambs made possible normal levels of oxygen delivery, which is a critical step toward the clinical application of artificial womb technology. ABSTRACT: EXTEND (EXTra-uterine Environment for Neonatal Development) is a novel system that promotes physiological development by maintaining the premature lamb in a sterile fluid environment and providing gas exchange via a pumpless arteriovenous oxygenator circuit. During the development of EXTEND, different cannulation strategies evolved with the aim of improving circuit flow. The present study examines how different cannulation strategies affect EXTEND circuit haemodynamics in extreme premature lambs. Seventeen premature lambs were cannulated at gestational ages 105-117 days (term 145-150 days) and supported on EXTEND for up to 4 weeks. Experimental groups were distinguished by cannulation strategy: carotid artery outflow and jugular vein inflow (CA/JV; n = 4), carotid artery outflow and umbilical vein inflow (CA/UV; n = 5) and double umbilical artery outflow and umbilical vein inflow (UA/UV; n = 8). Circuit flows and pressures were measured continuously. As we transitioned from CA/JV to CA/UV to UA/UV cannulation, mean duration of circuit run and weight-adjusted circuit flows increased (P < 0.001) and the frequency of flow interruptions declined (P < 0.05). Umbilical vessels generally accommodated larger-bore cannulas, and cannula calibre was directly correlated with circuit pressures and indirectly correlated with flow:pressure ratio (a measure of post-membrane resistance). We conclude that UA/UV cannulation in fetal lambs on EXTEND optimizes circuit flow dynamics and flow stability and also supports circuit flows that closely approximate normal placental flow.

The long non-coding RNA MALAT1 activates Nrf2 signaling to protect human umbilical vein endothelial cells from hydrogen peroxide.

The potential effect of the long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) against hydrogen peroxide (H2O2)-induced oxidative injury in endothelial cells was tested. We show that forced-expression of MALAT1 using a lentiviral vector ("LV-MALAT1") significantly attenuated H2O2-induced death and apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, knocking down of MALAT1 by targeted siRNA exacerbated H2O2-induced HUVEC injury. For the mechanism study, we show that LV-MALAT1 induced Keap1 downregulation, leading to nuclear-factor-E2-related factor 2 (Nrf2) stabilization and activation. Critically, Nrf2 shRNA almost completely abolished LV-MALAT1-mediated HUVEC protection against H2O2. Significantly, H2O2-induced oxidative stress, lipid peroxidation and DNA damages in HUVECs were attenuated by LV-MALAT1, but were intensified with MALAT1 siRNA. In summary, we identified a novel signaling axis involving MALAT1, Keap1 and Nrf2, which in turn protects HUVECs from oxidative injury.

Identification of Antiangiogenic Potential and Cellular Mechanisms of Napyradiomycin A1 Isolated from the Marine-Derived Streptomyces sp. YP127.

Angiogenesis is the process of new blood vessel formation. Excessive angiogenesis is a critical factor in the progression of cancer, macular degeneration, and other chronic inflammatory diseases. When investigating the effects of crude extracts of cultured marine microorganisms, an extract of the cultured Streptomyces sp. YP127 strain was found to inhibit human umbilical vein endothelial cell (HUVEC) tube formation. Bioassay-guided fractionation and spectroscopic data analyses led to the identification of napyradiomycin A1 (1) as an antiangiogenic component of the extract. Compound 1 inhibited HUVEC tube formation in a concentration-dependent manner. It inhibited endothelial cell proliferation but did not affect human dermal fibroblast proliferation. Compound 1 also suppressed migration and invasion of vascular endothelial cells. In addition, compound 1 suppressed vascular endothelial cadherin expression and increased the permeability of the endothelial cell membrane. These results suggested that compound 1 modulates cell permeability and inhibits the angiogenesis of endothelial cells.

[Relationships between umbilical vein and mother iron status].

INTRODUCTION: Iron is an essential micronutrient in the growing fetus. OBJECTIVE: The purpose of this study is to find the possible correlations that may exist between maternal and fetal iron status and newborn weight. MATERIAL AND METHODS: The study included 97 mothers scheduled to give birth by elective caesarean section in the central maternity of Tébessa (east of Algeria) between January and August 2014. The blood collection was sampled from the antecubital vein of the mother and the umbilical vein. The mean concentrations of parameters in maternal and fetal sides, respectively, were 10.64 ± 1.37 g/dl and 14.83 ± 1.79 g/dl for hemoglobin, 51.57 ± 20.82 µg/dl and 112.47 ± 32.34 µg/dl for serum iron, and 12.37 ± 9.58 ng/ml and 109.64 ± 58.76 ng/ml for serum ferritin. Except for ferritin, other fetal parameters were correlated with those of mothers. Birth weight was only significantly correlated with maternal hemoglobin (r = 0.22, p = 0.02) and hematocrit (r = 0.2, p = 0.004). CONCLUSION: The fetal-maternal exchanges of iron were highlighted and iron status of the newborn was linked to that of the mother. The low maternal hemoglobin was associated with low newborn weight.

Interleukin-33 induces growth-regulated oncogene-α expression and secretion in human umbilical vein endothelial cells.

Although interleukin-33 (IL-33), a member of the IL-1 cytokine family, plays proinflammatory roles in immune cells as an "alarmin," little is known regarding the biological actions of IL-33 on vascular endothelial cells. To investigate the effects of IL-33 on vascular endothelial cells, we first screened the IL-33-regulated proteins in human umbilical vein endothelial cells (HUVECs) using a dot blot array and observed that IL-33 markedly increased growth-regulated oncogene-α (GRO-α), a chemokine that is also known as chemokine (C-X-C motif) ligand 1 (CXCL1). Real-time reverse transcription PCR and ELISA demonstrated that IL-33 induced GRO-α expression and secretion in HUVECs in a dose- and a time-dependent manner. Western immunoblot assay revealed that IL-33 activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK). In addition, translocation of nuclear factor-κB (NF-κB) p65 to the nucleus of HUVECs was observed by IL-33 stimulation. Furthermore, treatment with pharmacological inhibitors against ERK1/2 (PD98059), JNK (SP600125), or NF-κB (BAY11-7085) significantly suppressed IL-33-induced GRO-α gene expression and secretion from HUVECs. Moreover, immunohistochemical staining demonstrated that IL-33 and GRO-α coexpressed in the endothelium of human carotid atherosclerotic plaque. Taken together, the present study indicates that IL-33 localized in the human atherosclerotic plaque increases GRO-α mRNA expression and protein secretion via activation of ERK1/2, JNK, and NF-κB in HUVECs, suggesting that IL-33 plays an important role in the pathophysiology and development of atherosclerosis.

Vasomotor effects of hydrogen sulfide in human umbilical vessels.

Hydrogen sulfide (H2S) has recently emerged as a biologically active gas with multiple effects on the cardiovascular system. We aimed to investigate the vasomotor actions of sodium sulfide (Na2S), which forms H2S and HS- in solution, in human umbilical artery (HUA) and vein (HUV) rings. In addition, we examined by immunocytochemistry the expression and localization of cystathionine ß-synthase (CBS), cystathionine lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (MPST), the enzymes responsible for endogenous H2S production. Human umbilical vessels were compared with chicken embryo umbilical vessels. HUA and HUV expressed a robust signal for CSE, CBS, and 3-MPST in both endothelial and smooth muscle cells. However, HUA rings did not respond to Na2S (10-6M-10-3M) either at resting tone or during contraction evoked by serotonin or KCl. Similarly, the extraembryonic part of chicken allantoic artery did not respond to Na2S. In contrast, Na2S induced a concentration-dependent contraction in HUV rings under resting tone and a concentration-dependent relaxation when the H2UV rings were contracted with serotonin (42 ± 5% relaxation) or KCl (12 ± 5% relaxation). Na2S-induced contraction of HUV was impaired following removal of extracellular Ca2+, endothelial denudation, NO synthase inhibition (L-NAME), or soluble guanylate cyclase (sGC) inhibition (ODQ). Na2S-induced relaxation of HUV was impaired by the KATP channel inhibitor glibenclamide. In conclusion, H2S does not have vasomotor effects on HUA but induced contraction (mediated through inactivation of the NO/sGC axis) and relaxation (mediated through KATP channels) in HUV. Our data suggest a role for H2S in the venous side of human umbilical circulation.

The structural and functional effects of fine particulate matter from cooking oil fumes on rat umbilical cord blood vessels.

A growing body of epidemiological evidence has supported the association between maternal exposure to airborne fine particulate matter (PM2.5) during pregnancy and adverse pregnancy outcomes. However, the specific biological mechanisms implicated in the causes of adverse pregnancy outcomes are not well defined. In this study, a pregnant rat model of exposure to different doses of cooking oil fumes (COFs)-derived PM2.5 by tail intravenous injection in different pregnant stages was established. The results indicated that exposure to COFs-derived PM2.5 was associated with adverse pregnancy outcomes, changed the structure of umbilical cord blood vessels, decreased the diameter and lumen area, and increased wall thickness. What's more, a significant increase of maximum contraction tension was observed in the early pregnancy high-dose exposure group and pregnant low-dose exposure group compared to the control group. Based on the maximum contraction tension, acetylcholine (ACh) did not induce vasodilation but caused a dose-dependent constriction, and there were significant differences in the two groups compared to the control group. Exposure to COFs-derived PM2.5 impaired the vasomotor function of umbilical veins by affecting the expression of NO and ET-1. This is the first study that evaluated the association of risk of adverse pregnancy outcomes and pregnant rats exposed to COFs-derived PM2.5 and primarily explored the potential mechanisms of umbilical cord blood vessels injury on a rat model. More detailed vitro and vivo studies are needed to further explore the mechanism in the future.

Progesterone suppressed vasoconstriction in human umbilical vein via reducing calcium entry.

The aim of this study was to evaluate the actions of progesterone on human umbilical vein (HUV) from normal pregnancies and the possible underlying mechanisms involved. HUV rings were suspended in organ baths and exposed to progesterone followed by phenylephrine (PE) or serotonin (5-HT). Progesterone suppressed PE- or 5-HT-induced vasoconstriction in HUV rings. The inhibitory effect induced by progesterone was not influenced by nitric oxide syntheses inhibitor, prostaglandins syntheses blocker, the integrity of endothelium, selective progesterone receptor or potassium channel antagonists. Further testing showed that progesterone and nifedipine (a blocker for L-type calcium channels) produced similar inhibitory effects on PE-, 5-HT-, Bay-k8644-, KCl-induced vasoconstriction in Krebs solution as well as CaCl2-induced vasoconstriction in Ca(2+)-free Krebs solution. But the inhibitory effect of mibefradil (mibe, a blocker for L-type (CaV1.2) and T-type calcium channels (CaV3.2)) on PE-, 5-HT-induced vasoconstriction was significantly greater than progesterone or nifedipine in Krebs solution. Furthermore, progesterone did not affect the vasoconstriction caused by PE, 5-HT, or caffeine in Ca(2+)-free Krebs solution. In addition, incubation HUV with progesterone did not change CaV1.2 and progesterone receptor (PR) expressions. The results gained demonstrated that progesterone could suppress multiple agonist-induced vasoconstrictions in HUV, mainly due to a reduction of calcium entry through L-type calcium channels, not endothelium-dependent vascular relaxation pathways, potassium channels, or Ca(2+) release from intracellular stores, providing new information important to further understanding the contribution of progesterone in the regulation of the placental-fetal circulation.

NF-κB signaling is essential for resistance to heat stress-induced early stage apoptosis in human umbilical vein endothelial cells.

Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

RTEF-1 protects against oxidative damage induced by H2O2 in human umbilical vein endothelial cells through Klotho activation.

Oxidative stress is a main risk factor of vascular aging, which may lead to age-associated diseases. Related transcriptional enhancer factor-1 (RTEF-1) has been suggested to regulate many genes expression which are involved in the endothelial angiogenesis and vasodilation. However, whether RTEF-1 has a direct role in anti-oxidation and what specific genes are involved in RTEF-1-driven anti-oxidation have not been elucidated. In this study, we found that overexpressing RTEF-1 in H2O2-treated human umbilical vein endothelial cells decreased senescence-associated-ß-galactosidase (SA-ß-gal)-positive cells and G0/G1 cells population. The expressions of p53 and p21 were decreased in H2O2-treated RTEF-1 o/e human umbilical vein endothelial cells. However, specific small interfering RNA of RTEF-1 totally reversed the anti-oxidation effect of RTEF-1 and inhibited RTEF-1-induced decreased p53 and p21 expressions. It demonstrated that RTEF-1 could protect cells from H2O2-induced oxidative damage. In addition, we demonstrated that RTEF-1 could up-regulate Klotho gene expression and activate its promoter. Furthermore, Klotho small interfering RNA significantly blocked RTEF-1-driven endothelial cell protection from H2O2-induced oxidative damage and increased p53 and p21 expressions. These results reveal that RTEF-1 is a potential anti-oxidation gene and can prevent H2O2-induced endothelial cell oxidative damage by activating Klotho.

Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells.

INTRODUCTION: Hypoxia induces dilatation of the umbilical vein by releasing autocoids from endothelium; prostaglandins (PGs), adenosine and nitric oxide (NO) have been implicated. ATP is vasoactive, thus we tested whether hypoxia releases ATP from primary Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were grown on inserts under no-flow conditions. ATP was assayed by luciferin-luciferase and visualised by quinacrine labeling. Intracellular Ca(2+) ([Ca(2+)]i) was imaged with Fura-2. RESULTS: ATP release occurred constitutively and was increased by hypoxia (PO2: 150-8 mmHg), ∼10-fold more from apical, than basolateral surface. Constitutive ATP release was decreased, while hypoxia-induced release was abolished by brefeldin or monensin A, inhibitors of vesicular transport, and LY294002 or Y27632, inhibitors of phosphoinositide 3-kinases (PI3K) and Rho-associated protein kinase (ROCK). ATP release was unaffected by NO donor, but increased by calcium ionophore, by >60-fold from apical, but <25% from basolateral surface. Hypoxia induced a small increase in [Ca(2+)]i compared with ATP (10 µM); hypoxia inhibited the ATP response. Quinacrine-ATP fluorescent loci in the perinuclear space, were diminished by hypoxia and monensin, whereas brefeldin A increased fluorescence intensity, consistent with inhibition of anterograde transport. DISCUSSION: Hypoxia within the physiological range releases ATP from HUVEC, particularly from apical/adluminal surfaces by exocytosis, via an increase in [Ca(2+)]i, PI3K and ROCK, independently of NO. We propose that hypoxia releases ATP at concentrations sufficient to induce umbilical vein dilation via PGs and NO and improve fetal blood flow, but curbs amplification of ATP release by autocrine actions of ATP, so limiting its pro-inflammatory effects.

Theranostic Gold Nanomicelles made from Biocompatible Comb-like Polymers for Thermochemotherapy and Multifunctional Imaging with Rapid Clearance.

A new generation of photothermal theranostic agents based on assembling 6 nm gold nanoparticles (AuNPs) is developed by using a novel comb-like amphipathic polymer as the template. The small AuNPs are assembled into DOX@gold nanomicelles, which show strong absorbance in the near-infrared region, for multimodal bioimaging and highly effective in vivo chemotherapy and photothermal therapy.

Measurement of the circumferential mechanical properties of the umbilical vein: experimental and numerical analyses.

Coronary artery disease is responsible for almost 30% of all deaths worldwide. The saphenous vein and umbilical vein (UV) are the most common veins using for treatment as a coronary artery bypass graft (CABG). The mechanical properties of UV belonging to its long-term patency for CABG are very important. However, there is a lack of knowledge on the linear elastic and nonlinear hyperelastic mechanical properties of the UV. In this study, three stress definitions (second Piola-Kichhoff stress, engineering stress and true stress) and four strain definitions (Almansi-Hamel strain, Green-St Venant strain, engineering strain and true strain) are used to determine the elastic modulus, maximum stress and strain of eight human UVs under circumferential loading. The nonlinear mechanical behaviour of the UV is computationally investigated using Mooney-Rivlin hyperelastic model. A numerical finite element analysis is also carried out to simulate the constitutive modelling versus its numerical results. The results show that the Almansi-Hamel strain definition overestimates the elastic modulus while Green-St Venant strain definition underestimates the elastic modulus at different stress definitions. The true stress-true strain definition, which gives more accurate measurements of the tissue's response using the instantaneous values, reveals the Young's modulus and maximum stress of 2.18 and 6.01 MPa, respectively. The Mooney-Rivlin material model is well represented by the nonlinear mechanical behaviour of the UV. The findings of this study could have implications not only for understanding the extension and rupture mechanism of UV but also for interventions and surgeries, including balloon angioplasty, bypass and stenting.

Are 1st-trimester ß-human chorionic gonadotrophin and pregnancy-associated plasma protein A levels predictive of intrapartum fetal compromise in a selected normal population?

BACKGROUND: The incidence of cerebral palsy in term infants has not changed over the last 30 years. Current intrapartum monitoring techniques are limited by their inherent poor specificity. Changes in fetal haemodynamics in the term fetus, similar to those seen in fetal growth restriction, have been associated with an increased risk of subsequent intrapartum fetal compromise. Alterations in first-trimester ß-hCG and PAPP-A levels are predictive of fetal growth restriction. AIMS: In this study, we aimed to establish whether first-trimester ß-hCG and PAPP-A levels were predictive of fetal compromise in labour and whether these first-trimester markers could be correlated with fetal haemodynamics at term in a low-risk population. MATERIALS AND METHODS: Over a two-year period, 427 women with low risk, uncomplicated pregnancies were recruited to this study. All participants underwent a prelabour ultrasound examination during which fetal biometry and haemodynamics were assessed. First-trimester ß-hCG and PAPP-A levels were recorded from the case notes. All cases were followed up within 48 hours of delivery, and first-trimester ß-hCG and PAPP-A levels correlated with intrapartum outcomes and fetal haemodynamics. RESULTS: No significant relationship between first-trimester ß-hCG and PAPP-A levels and subsequent intrapartum fetal compromise was observed. Weak but significant correlations were observed between ß-hCG levels and umbilical venous flow rate, as well as PAPP-A levels and uterine artery pulsatility index. CONCLUSIONS: ß-hCG and PAPP-A levels measured during the first trimester are not predictive of subsequent intrapartum fetal compromise within a low-risk population.

Click chemistry plays a dual role in biodegradable polymer design.

Click chemistry plays a dual role in the design of new citrate-based biodegradable elastomers (CABEs) with greatly improved mechanical strength and easily clickable surfaces for biofunctionalization. This novel chemistry modification strategy is applicable to a number of different types of polymers for improved mechanical properties and biofunctionality.

Ductus venosus closure results in transient portal hypertension--is this the silent trigger for necrotizing enterocolitis?

INTRODUCTION: The etiology of necrotizing enterocolitis (NEC) remains elusive and no definite trigger has been identified. There are no studies to date examining the potential role of closure of the ductus venosus (DV), its effect on increasing portal venous pressure (PVP) and its association to mesenteric venous ischemia in the development of NEC. Our aim was to develop an animal model to examine this physiology. METHODS: Fifteen near-term lambs were used. The DV was occluded in experimental animals by a balloon tip catheter, while the sham controls underwent catheterization without DV occlusion. Vital signs and PVP were monitored for 4h, followed by intestinal biopsy. RESULTS: The experimental group (n=5) demonstrated a significant increase in PVP following DV occlusion (11.87 mm Hg [95% CI: 11.40-12.34]), compared to controls (8.95 mm Hg [95% CI: 8.34-9.56]) (F=12.16, p=0.001). Histology of the terminal ileum showed vacuolar degeneration, indicative of reversible cellular damage in the experimental group. CONCLUSIONS: We demonstrate that DV closure in the neonatal lamb leads to transient portal hypertension which is associated with cellular damage and inflammatory changes of the intestinal mucosa. Additional studies will be necessary to determine if the transient portal hypertension following DV closure leads to clinically apparent intestinal ischemia and NEC.

Vasopeptidase-activated latent ligands of the histamine receptor-1.

Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 µM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 µM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases.

Co-culture of mesenchymal stem cells with umbilical vein endothelial cells under hypoxic condition.

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdM(NOR), CdM-CdM(HYP) and HUVEC-CdM(HYP) were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdM(NOR) and HUVEC-CdM(HYP) groups, the survival rate, migration and angiogenesis of HUVECs in MSC-CdM(HYP) group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdM(HYP) group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.