In utero exposure of delta-9-tetrahydrocannabinol (THC) affects vascular transcriptome in rhesus macaques
Cannabis is commonly used in pregnancy for symptoms of nausea and pain, especially in the first trimester. Cannabis remains a federally illicit drug in the United States, but local legalization trends have resulted in increased availability and a decreased perception of harm. Unfortunately, limited scientifically rigorous information exists to inform decisions on use. Delta-9-tetrahydrocannabinol (THC, the main psychoactive component of cannabis) can cross the placenta and bind to cannabinoid receptor 1 (CB1) on the fetus. Because CB1 receptors are expressed in cardiomyocytes and endothelial cells, this suggests that THC exposure may impact fetal cardiovascular development. To understand this impact, our group used an established rhesus macaque model of chronic edible THC use during pregnancy. Animals were slowly titrated to a heavy THC dose (2.5mg/7kg/day) for 4 months preconception. Dams continued this daily THC dose throughout pregnancy with c-section delivery near term. Our model showed a decreased heart-to-body weight ratio from THC exposure. We examined the underlying changes in this selective fetal growth restriction of the cardiovascular system through tissue, protein, and gene expression analyses. H istological analysis of coronal sections of the heart and cross sections of the aorta showed no changes in collagen expression or maturity. Western blot analysis of collagen III expression showed no changes in left or right ventricle. Elastin expression was also unchanged in the aorta. To assess transcriptional changes, we performed bulk RNA-sequencing of vascular cells in the fetal aorta, umbilical vein, and umbilical artery, which revealed differentially expressed genes involved in metabolism and inflammation.  These results suggest potential cardiovascular harm from in utero THC exposure by altering endothelial metabolism and inflammation. Overall design: Indoor-housed female rhesus macaques (n = 10) were maintained on a standard chow diet (TestDiet). Animals were given a daily cookie containing THC made with research-grade THC obtained directly from the National Institute of Drug Abuse (NIDA). Animals were slowly titrated up to 2.5 mg/7 kg/day of THC approximately four months prior to conception. Animals (n=5) continued daily THC edible throughout pregnancy until cesarean section delivery at gestational day 155. Fetal necropsy was performed and heart and umbilical cord tissues were processed as described in detail below. Aorta and umbilical cord vessels were harvested and sectioned for multiple use processing. Three centimeters of each vessel, in duplicate, was cannulated and flushed with 1X PBS (Gibco) to remove blood followed by 100uL of TRIzol Reagent (Invitrogen) to lyse luminal cells and collect RNA. The effluent was immediately frozen on dry ice and stored at -70oC.