No abstract

Raising infant piglets in an environment of 10% oxygen for 4 weeks results in significant pulmonary arterial hypertension accompanied by increased expression of nitric oxide synthase within the lung endothelium. Furthermore, the increased levels of nitric oxide synthase within the lungs of the hypoxic swine were not accompanied by a proportional increase in enzyme activity. These findings suggest that the development of pulmonary hypertension in infants with congenital cyanotic disease is not due to decreased expression of endothelial nitric oxide synthase, but instead may be related to a decreased ability of the enzyme to produce sufficient nitric oxide.

An optimized assay for alkaline phosphatase (EC 3.1.3.1) is reported. A discrete analyzer, the DuPont Automatic Clinical Analyzer (aca), was used for this study. The assay is based on results of response-surface experimental co-optimization techniques, and response is enhanced over the present aca assay. A key feature is the incorporation of zinc ions, both to fully optimize the assay and to reduce the sensitivity of measured activity to zinc-binding impurities in the buffer, 2-amino-2-methyl-1-propanol. In addition, a simple technique is described for measuring relative concentrations of zinc-binding impurities in this buffer. These features should be considered in the design of any assay for alkaline phosphatase that is based on p-nitrophenyl phosphate as substrate and 2-amino-2-methyl-1-propanol as buffer.

Background Macroautophagy is a highly conserved cellular process in which cytoplasmic materials are internalized into an autophagosome that later fuses with a lysosome for their degradation and recycling. MicroRNAs (miRNAs) are integral regulators in a variety of cellular processes and are known to regulate autophagy, and miRNA and autophagy both play roles in the regulation of endothelial function. Accordingly, we hypothesize that miRNA miR‐378‐3p is an essential regulator of endothelial autophagy and endothelial function. Methods and Results To test our hypothesis, we cultured human umbilical vein endothelial cells (HUVEC) and confirmed the basal expression of miR‐378‐3p. To determine the effect of autophagy on miR‐378a‐3p, autophagy was genetically (via silencing autophagy‐related gene ATG7) and pharmacologically (via chloroquine treatment) inhibited in HUVECs, which resulted in the upregulation of miRNA miR‐378a‐3p. We also measured miR‐378a‐3p expression following autophagy activation (via starvation) and observed a significant down‐regulation of miR‐378‐3p expression. Next, we over‐expressed miR‐378a‐3p (by mimic) in HUVECs and measured autophagy and endothelial function in the form of endothelial cell proliferation and migration. MiR‐378a‐3p over‐expression was associated with impaired autophagy indicated by reduced LC3‐II/LC‐3‐I ratio, reduced proliferation and migration in HUVECs. At the molecular level, miR‐378a‐3p over‐expression was associated with increased mTOR (mammalian target of rapamycin; an autophagy regulator) expression at the transcript and the protein levels in HUVECs. Conclusion Our preliminary findings indicate an essential role of miR‐378a‐3p in the regulation of endothelial autophagy and endothelial function. We demonstrate an inverse relationship between miR‐378a‐3p expression and endothelial autophagy and function, warranting future investigations.

While it has been well established that the development of metabolic disease, including hypertension, impaired glycemic control, etc. can be associated with increased myogenic activation of cerebral resistance arteries and arterioles, the impact of this shift in pressure‐induced responses on integrated vascular function remains elusive. Using male obese Zucker rats (OZR) at 17–18 weeks of age, we determined that changes to myogenic activation of ex vivo pressurized middle cerebral arteries (MCA) was best predicted by the severity of elevation in mean arterial pressure and allowed for stratification into three groups (mild, moderate, high degrees of elevation in the slope of myogenic activation vs. intravascular pressure). While dilator responses to endothelium‐dependent stimuli (e.g., acetylcholine) were attenuated in all MCA of OZR, the severity of the attenuation was correlated with the degree of increase in myogenic activation from normal for that vessel. Treatment of the vessels with the anti‐oxidant TEMPOL significantly improved vascular responses to acetylcholine in the mild and moderate myogenic activation groups of MCA from OZR, but had minimal impact on high myogenic response groups. In contrast, treatment of vessels with the mechanosensitive cation channel inhibitor gadolinium chloride to reduce myogenic activation in OZR MCA to levels comparable to that of control LZR had minimal impact on endothelial function in MCA from the mild and moderate groups, but significantly improved endothelium‐dependent responses in MCA from the high group. Combined treatment of OZR MCA with both gadolinium chloride and TEMPOL resulted in endothelium‐dependent responses being very comparable across groups. Assays for vascular nitric oxide and PGI2 bioavailability provided correlative evidence that endothelial function, while still impaired in OZR in general, may be better maintained in vessels from animals with a higher myogenic activation than would have otherwise been predicted. Calculations suggest that the increased myogenic activation in the “high” group may serve to reduce wall tension in response to the elevated arterial pressure and that this may contribute to a mechanism that partially protects endothelial function. Support or Funding Information CIHR #389769; NSERC RGPIN‐2017‐04536 and RGPIN‐2018‐05450