Hydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.

-Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of largeconductance Ca 2ϩ -activated K ϩ (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca 2ϩ . Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca 2ϩ signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (ϳ140 days gestation) and newborn, 10-to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for Ͼ100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca 2ϩ responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 M nitro-L-arginine methyl ester or 10 M 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel ␣1-subunit expression but increased ␤1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes. potassium channels; sheep; pulmonary artery; contractility; maturation; hypoxia REGULATION OF SMOOTH MUSCLE tone in pulmonary arteries during development is a delicate balance of vasoconstrictive and vasorelaxant pathways. Endothelial cells play a crucial role in determining the overall level of vasorelaxation (39, 67), and endothelium-dependent relaxation is partially mediated through bradykinin stimulation (31). Bradykinin is a potent vasodilator that is important in the fetal pulmonary circulation, as well as during inflammation, and its relationship to pulmonary hypertension has been explored (5, 31, 83).Endothelial bradykinin receptor activation induces vasorelaxation through modulation of several different intracellular signaling pathways that are largely dependent on a rise of endothelial intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (67). The most widely studied pathway is bradykinin-induced activation of endothelial nitric oxide (NO) synthase (eNOS), an enzyme that generates NO (64). NO acts on nearby smooth muscle cells to cause downstream stimulation of soluble guanylate cyclase (sGC) pathways that leads to vasorelaxation (3,45). Previous studies have shown that regulatio...

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca channels (Ca), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca oscillations, and ryanodine receptor-driven Ca sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K, FPL contractions were greater in fetuses than in adults. FPL reduced Ca oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that Ca-dependent contractions are largely uncoupled from intracellular Ca oscillations and the development of Ca sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant Ca facilitation, and the related associations with the activation of Ca oscillations and Ca sparks.

Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human U937 cell adhesion to NO-deficient human microvascular ECs in vitro through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamellar lipid vesicles (DPhPC, POPC, Cholesterol, DHPE-Texas Red at molar ratio of 6:2:2:0.2) that were ~155 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced a 70-fold increase in NTG therapeutic efficacy when compared with free NTG while preventing excessive mitochondrial superoxide production associated with high NTG doses. Thus, these findings, which are the first to reveal the superior therapeutic effects of an NTG nanoformulation, provide the rationale for their detailed investigation for potentially superior vascular normalization therapies.

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI), and large-conductance K (BK) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca with confocal microscopy. eNOS was inhibited with 100 μM N-nitro-l-arginine methyl ester (l-NAME), PGI production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BK channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca signals and bradykinin relaxation. The Ca ionophore A-23187 (10 µM) elicited an enhanced Ca response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BK function decreased during bradykinin relaxation following LTH. Western analysis showed that BK α-subunit expression was increased by LTH while that for the β subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.