Prenatal Morphine Exposure Increases Cardiovascular Disease Risk and Programs Neurogenic Hypertension in the Adult Offspring

Abstract

Background:

The opioid overdose and opioid use disorder (OUD) epidemics are concomitant with increased metabolic and CVD risk. Although OUD causes adverse pregnancy outcomes, the offspring’s cardiovascular health is understudied. We hypothesized that offspring exposed to morphine (MOR) in utero would show increased CVD risk factors and endogenous opioid system dysregulation.

Methods:

Sprague Dawley dams were treated with saline (VEH, n=10) or escalating doses of MOR (5-20mg/kg/day, s.c, n=10) during gestation. Cardiovascular and metabolic parameters were assessed in adult offspring.

Results:

Litter size and pups’ birth weight were not different in response to MOR exposure. Female and male MOR-exposed offspring showed reduced body length at birth (p<0.05) and body weight from weeks 1-3 of life (p<0.05), followed by a catch-up growth effect. By week 16, female and male MOR-exposed rats showed reduced tibia length (p<0.05) and fat mass. in utero MOR exposure (IUME) increases the mean arterial pressure and the depressor response to mecamylamine (5mg/kg/day, i.p) were abolished by a chronic treatment with an alpha-adrenergic receptor blocker (prazosin, 1mg/kg/day, i.p.). Although circulating levels of angiotensin peptides were similar between groups, IUME exacerbated maximal ex vivo AngII-induced vasoconstriction (p<0.05) and induced endothelial dysfunction in a sex-specific manner (p<0.05). Proenkephalin, an endogenous opioid peptide that lowers blood pressure and sympathetic-mediated vasoconstriction, showed reduced mRNA expression in the heart, aorta, and kidneys from MOR vs. VEH group (p<0.05).

Conclusion:

Among the effects of IUME, neurogenic hypertension, vascular dysfunction, and metabolic dysfunction could be associated with the dysregulation of the endogenous opioid system.

Graphical Abstract

Introduction

Opioid misuse, OUD, and opioid overdoses are great public health threats in the US^1,2. From 2019 to 2020, drug overdose deaths increased by 31%, with 75% of these deaths involving opioids¹. In 1999, an initial opioid overdose death wave was caused by prescription opioids, both natural and semisynthetic opioids (e.g., morphine and oxycodone), followed by the second wave of deaths in 2010 caused by heroin (semisynthetic opioid), which is metabolized to MOR ¹. However, ‘a third concerning wave caused by synthetic opioids, specifically illicit fentanyl and fentanyl-like analogs, has been on an alarming rise since 2013 ¹. Nevertheless, an understudied area related to the opioid epidemic is OUD during pregnancy, which results in a 5-fold increase in neonatal opioid withdrawal syndrome (NOWS) incidence^2–4, a group of symptoms affecting different body systems (e.g., cardiovascular, gastrointestinal). NOWS severity is influenced by the length of exposure, type of opioid, and other licit and illicit substances that the mother uses. Strikingly, every hour four babies are diagnosed with this syndrome ^2,3,5.

A growing number of studies shed light on possible adverse effects on health outcomes associated with long-term opioid use ^6–8. The hospitalization trends due to cardiovascular events mirror trends of opioid overdose⁹. In this sense, OUD is associated with a 2-fold increased risk of hypertension, myocardial infarction, and ischemic heart disease^10–12. OUD is associated with an increased risk of a metabolic-like syndrome¹³, worsening of preexisting diabetes condition by impairing glucose metabolism, promoting insulin resistance, altering lipid profile^14–16, atherogenesis, and coronary artery disease^11,14,15,17, all major risk factors for CVD. Nevertheless, there are significant gaps in the scarce literature investigating the mechanisms underlying the interaction between opioids and systems modulating cardiovascular function. The current gap in assessing CVD risk in the offspring of women experiencing OUD during pregnancy raises a concern regarding the opportunity to identify a group of individuals at risk and improve the current therapeutic approaches to prevent adverse health outcomes ^3,18.

A critical point in the decision-making to treat OUD is understanding the interaction between different types of opioid agonists and antagonists with opioid receptors. The endogenous opioid system (EOS) comprises endogenous opioid peptides (EOPs) and five opioid receptors (ORs): µ -OR, κ-OR, δ-OR, nociception-OR, and z-OR, expressed in most tissues, including the brain, heart, kidneys, liver, and intestinal tract¹⁹. ORs are G-protein coupled receptors (GPCRs) that can dimerize and affect the function of other GPCRs involved in cardiovascular regulation^20–22. For instance, opioids, angiotensin II, and adrenergic receptors are GPCRs expressed within brain areas responsible for blood pressure control and sympathetic outflow, such as the paraventricular nucleus of the hypothalamus (PVN), the rostroventrolateral medulla (RVLM), and the nucleus of the solitary tract (NTS)^23–26. Moreover, the peripheral expression of ORs subtypes in the vasculature have been shown to play a role in endothelial function by impairing or improving vascular relaxation upon the activation of mu-OR or K-OR, respectively ^27–29.

While numerous preclinical studies of prenatal opioid exposure have been used to investigate long-term consequences in behavior outcomes, analgesia, reward, learning, and memory processing, only a handful of studies have assessed the detrimental effects of opioid use on cardiovascular function^12,29–31. Moreover, the study of potential sex differences secondary to prenatal opioid exposure has been lacking altogether³¹. Consequently, there is a remarkable gap in the knowledge addressing the link between opioid exposure during developmental life and long-term cardiovascular risk^3,18.

This study aimed to develop a rat paradigm of opioid exposure during pregnancy using MOR, the prototypic μ-OR agonist, to assess the long-term cardiovascular effects on the offspring. The ultimate goal of this study is to use this model as a tool to investigate molecular mechanisms, identify targets, and test therapeutic approaches to reduce the effects of opioid exposure increasing cardiovascular risk factors in the offspring. Thus, we investigated litter morphometrics, body composition, blood pressure, and sympathetic activation with and without the effects of a chronic alpha adrenergic receptor blocker, blood and urine biomarkers of cardiovascular and sympathetic function, and the effect of ORs on vascular function in male and female offspring exposed to MOR in utero. In addition, we determined PENK expression in cardiovascular tissues and the locomotor sensitization to MOR exposure in adulthood to assess potential neurobehavioral alterations following in utero MOR exposure (IUME).

Methods

The data supporting the findings of this study are available upon reasonable request from the corresponding author. Detailed protocols can be found in the Supplementary Material section.

Animals.

All experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved and monitored by the Institutional Animal Care and Use Committee at the University of Kentucky. Sprague Dawley female and male rats used for breeding had ad libitum access to food and water and were housed in a pathogen-free environment with constant temperature and humidity, with a 12:12 hour light: dark cycle. Animals were fed a regular chow diet consisting of 24% protein, 58% carbohydrates, and 18% fat with 3.1 kcal/g gross energy (Teklad 8604, Madison, WI).

Morphine exposure during pregnancy.

Sprague-Dawley rats (250–300 g) were bred in-house. A vaginal smear for timed pregnancy was performed between 8:00 and 9:00 AM for two weeks on female breeders. Two days prior to mating, bedding of the male was introduced in the female cage and males were added when the females were in the proestrus phase. Gestational day 0 (G0) was determined by the presence of a sperm plug. Once GD0 was determined, dams were randomly assigned to morphine (MOR, n=10) and vehicle (VEH, n=10) groups. Dams were treated with a daily subcutaneous injection of VEH (sterile 0.9% NaCl) or an escalating dose of morphine sulfate GD1-3 (5mg/kg/day, manufactured by Hospira Laboratory, supplied by Henry Schein using DEA registration number RL0457730). This dose was increased by 5 mg/kg/day every five days from GD4-GD19 to reach the final dose of 20mg/kg/day. At birth, litters were culled to 8-10 pups and balanced for sex. Body weight, composition, and morphometrics data were averaged per litter. For functional tests, one adult offspring per sex per litter was randomly assigned to different experiments.

Experimental design.

Weight gain during pregnancy and lactation was assessed every other day. At birth, body weight and morphometric measurements (head and waist circumference, anogenital distance, tail length, total body length) were taken in all pups during the first 8 hours of life. At weaning, body composition was determined using the Echo Magnetic Resonance Imaging system (Echo-MRI; Echo Medical Systems, Houston, TX). Then, body weight was assessed weekly. At 14 weeks, rats were placed in metabolic cages for 24-hour acclimation. Then, food and water intake and urine output were measured for two consecutive days. A week later, a magnetic resonance imaging (MRI) scan was performed to determine body composition^32–34. In a second subset of 16-week old offspring, fasting blood glucose and metabolic function were determined^35,36, allowing rats to recover for a few days before the endpoint euthanasia with ketamine/xylazine (100/10mg/kg, IP) for blood collection, tissue harvesting for gene expression³⁷ and vascular function studies³⁸. In a third subset of rats, direct, conscious blood pressure was measured in untreated or treated with an α-adrenergic receptor blocker (prazosin, 1mg/kg, i.p., 2 weeks). In the last subset of rats, a locomotor test was performed³⁹ following repeated exposure to morphine (10mg/kg/day) for 6 consecutive days.

Plasma and serum biomarkers.

Fasting blood glucose was determined by analyzing a drop of blood using a glucometer (Accu-Check, Roche, Germany). ELISA kits were used to measure plasma insulin (Invitrogen by Thermo Fisher Scientific, Waltham, MA) and leptin (Cayman, Ann Harbor, MI). Serum renin-angiotensin system (RAS) fingerprints were determined at the University of Kentucky RAS Analytical Lab Service Center^40–42. Serum cholesterol and lipoproteins were determined using fast protein liquid chromatography (FPLC)^43,44 and plasma and Urine Norepinephrine (NE) were measured by modifications of HPLC protocols^45,46.

Conscious measurement of blood pressure.

Rats were fitted with femoral artery cathethers as previously reported.³⁶ The following morning, rats were awake and freely moving in the housing cage, acclimated for 2 hours, and a baseline blood pressure was measured for 2 hours under continuous monitoring (PowerLab, ADInstruments). Acute angiotensin II (AngII) intraarterial boluses (0.01-1.2 nMol/Kg) were performed randomly during baseline. Thirty minutes after the last AngII dose, a bolus of the ganglion blocker mecamylamine (5mg/kg, i.p.) was performed to determine changes in blood pressure over 1-hour period.

Statistical Analysis.

All data are presented as mean±SEM. Comparison between VEH and MOR was assessed via t-test. When sex was an added factor, a 2-way ANOVA analysis was performed. Data for body composition trajectories were analyzed by 2-Way ANOVA repeated measures followed by Tuckey multiple comparisons post-hoc test for body composition trajectories. Detailed statistic tests are provided in the legend of each figure. Analyses were performed using GraphPad Software version 9.0.0 (La Jolla, CA, www.graphpad.com). Statistical significance was determined by p<0.05.

Results

Prenatal MOR exposure leads to smaller offspring that experience a catch-up growth effect.

Dams treated with escalating doses of MOR showed a similar increase in weight gain during pregnancy compared to VEH-treated dams (26.5±2.5% versus 26±3.8% of BW). The litter size, number of female and male pups (Fig. 1A), and birth weight (Fig. 1B) were similar between groups. Morphometric measurements showed no differences in anogenital distance, head circumference, waist circumference, cephalization index, and tail length between VEH and MOR-exposed offspring. On the contrary, body length (nose to anus) and full length (nose to last caudal vertebra) were significantly reduced in male and female IUME offspring (Fig. 1C–D). Further, IUME offspring showed reduced BW from week 1 to week 3; however, both groups showed similar fat and lean mass composition at weaning (Fig.S1). MOR-exposed females (Fig. 1E) and males (Fig. 1F) experienced catch-up growth by week 4, showing similar BW between VEH and MOR groups until week 16.

Figure 1. Morphine exposure does not affect pregnancy outcomes however results in catch-up growth in the in utero morphine exposed offspring:

A) total litter size B) female and male neonates birth weight, C) morphometric measurements in female neonates, D) morphometric measurements in male neonates, E) body weight trajectory from weeks 1-16 in female offspring, F) body weight trajectory from weeks 1-16 in male offspring.

Data were averaged by litter, analyzed by t-test and reported as mean ± SEM. Data were analyzed by 2-Way ANOVA repeated measures followed by Tuckey multiple comparisons post-hoc test for body composition trajectories; n=9-10/group; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, PD=postnatal day, AGD=anogenital distance, WC=waist circumference, HC=Head circumference, TL=tail length FL=full length, CI=cephalization index

Organs and tissues weights were not influenced by MOR exposure in female rats (Fig. 2A), while male rats showed a significant reduction in subcutaneous and retroperitoneal white adipose tissue fat pads (Fig. 2B). Tibia length was reduced in IUME female and male adult rats (Fig. 2A–B insets).

Figure 2. In utero morphine exposure reduces fat mass in adult offspring:

A) tissue and organ weights and tibia length (inset graph) in adult female rats, B) tissue and organs weights and tibia length (inset graph) in adult male rats, C) representative MRI scan of adult female rats, D) representative MRI scan of adult male rats, E) quantification of the subcutaneous and visceral fat in female rats, F) quantification of the subcutaneous and visceral fat in male rats.

Data were averaged by litter, analyzed by t-test and reported as mean ± SEM; n=6-8/group for tissue, organs weight and tibia length and MRI analysis; * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, gWAT=perigonadal white adipose tissue, scWAT= subcutaneous white adipose tissue, periWAT=perirenal white adipose tissue, SC=subcutaneous, VISC=visceral, MESE=mesenteric white adipose tissue, BAT=brown adipose tissue.

MRI full body scans from female rats (Fig. 2C) showed a similar amount of adipose tissue, as the quantification of visceral and subcutaneous fat proportional to BW was similar between groups (Fig. 2E). However, IUME male rats displayed reduced subcutaneous and visceral fat depots proportional to BW (Fig.2D and 2F).

Prenatal MOR exposure is associated with increased circulating cardiovascular risk biomarkers.

IUME adult rats showed increased fasting blood glucose (FBG) (Fig. 3A), reduced plasma insulin (Fig. 3B) and leptin (Fig. 3C). IUME did not change water and food intake and urinary excretion in female or male rats (Table S2). Lipid profile showed IUME increased low-density lipoproteins (LDL) in female and male rats (Fig. 3D). Despite IUME increased albumin to creatinine ratio similarly in both sexes (Figure 3E), only IUME females showed a reduced creatinine clearance compared with vehicle (Fig.S2). Plasma and urine NE were increased in male rats exposed to prenatal MOR (Fig. S3 and 3F, respectively).

Figure 3. In utero morphine exposure increases plasma and urine biomarkers of cardiometabolic disease risk factors:

A) FBG, B) insulin, C) leptin, D) FPLC analysis of plasma male and female rats, E) ACR and F) urinary NE.

Data were analyzed by 2-Way ANOVA followed by Tuckey multiple comparisons post-hoc test and reported as mean ± SEM; n=8-10/group, Fig 3A–F,F, n=6-8/group, Fig. 3G–H;H; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, FBG=fasting blood glucose, ACR= albumin creatinine ratio, NE= norepinephrine.

Prenatal MOR exposure increases blood pressure and sympathetic tone.

IUME induced increases in MAP were abolished by the prazosin treatment (Fig. 4A). Furthermore, mecamylamine induced a significant reduction in MAP in IUME female rats compared to their VEH counterparts. Prazosin abrogated this reduction (Fig. 4B). IUME male rats showed the same increase in MAP and significant reduction in response to mecamylamine compared with VEH. The chronic treatment with prazosin reduced blood pressure at baseline and reduced mecamylamine depressor response in IUME male rats (Fig. 4C and 4D). When sex was analyzed as a factor, IUME did not worsen baseline blood pressure or degree of sympathetic tone (not shown). Overall, the expression of adrenergic receptors were downregulated in most of the tissues analyzed. Specifically, MOR-exposed females showed reduced mRNA expression of αA1-AR in heart, αB1 and αB2-AR in renal outer medulla (OM), and β2-AR in renal cortex and OM. Also αB2-AR was increased in heart compared to VEH female rats (Fig. 4E). In males, MOR-exposed offspring showed reduced mRNA expression of αA1-AR in aorta, heart, and renal OM, αB1-AR in aorta and heart, αB2-AR in renal OM and β2-AR in aorta and OM (Fig. 4F).Circulating levels of RAS components were similar between groups of both sexes (Fig. S3A and B). However, the delta dose response to acute doses of AngII was significantly attenuated in MOR-exposed female rats only (Fig. S3C) while showing a subtle effect on the male rat (Fig. S3D).

Figure 4. In utero morphine exposure increases blood pressure and sympathetic activation:

A) Mean arterial pressure in female rats untreated and treated with prazosin, B) acute blood pressure response to mecamylamine in female rats untreated and treated with prazosin, C) mean arterial pressure in male rats untreated and treated with prazosin D) acute blood pressure response to mecamylamine in female rats untreated and treated with prazosin E) cardiovascular tissue adrenergic receptor expression in female rats, and F) cardiovascular tissue adrenergic receptor expression in male rats. Data were analyzed by 2-Way ANOVA followed by Tuckey multiple comparisons post-hoc test for blood pressure in A and B. For AngII-induced pressor response, data were analyzed using a 2-way ANOVA repeated measures, while RAS components were analyzed by t-test. Data were reported as mean ± SEM; n=7-9/group * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, MAP=mean arterial pressure, Meca=mecamylamine, PRAZ=prazosin.

Prenatal MOR exposure induces a sex-specific impairment of vascular function.

The AngII-induced response in isolated rings tended to increase in MOR-exposed female offspring while constricted similarly with and without naloxone pre-incubation (Fig. 5A). Conversely, rings from MOR-exposed male rats displayed increased AngII-induced vasoconstriction that was abrogated by naloxone treatment (Fig. 5B). The mRNA expression for AT1 and ACE was significantly reduced in aortas from MOR-exposed offspring compared to VEH (Fig. S5).

Figure 5. In utero morphine exposure promotes sex-specific impairments of the vascular function:

AngII-induced constriction with and without naloxone in rings from A) female and B) male rats, C) PE-induced constriction in male and female rats, D) KCl-induced constriction in male and female rats, Ach and SNP-induced relaxation in rings from E) female and F) male rats.

Data were reported as mean ± SEM; n=7-9/group * p<0.05 vs. control. VEH=vehicle, MOR=Morphine, F=females, M=males, MAP=mean arterial pressure, AngII=angiotensin II, PE=phenylephrine, Ach=acetylcholine, SNP=sodium nitroprusside.

PE-induced constriction (Fig. 5C) and the maximal response to KCl in isolated aortas (Fig. 5D) were similar in MOR and VEH-exposed groups in both sexes. Moreover, the endothelial function was impaired in IUME females only, as evidenced by the reduced Ach-induced relaxation (Fig. 5E–F), whereas the receptor-independent SNP-induced relaxation was similar between groups in both sexes.

Prenatal MOR exposure reduces the expression of an endogenous opioid peptide in vascular tissues.

PENK mRNA levels were reduced in the aorta and kidney (cortex and OM) of IUME females (Fig. 6A) and male rats (Fig. 6B). IUME induced a reduction in PENK only in the heart of male rats. Conversely, PENK expression was significantly increased in the liver of IUME adult offspring.

Figure 6. In utero morphine (MOR) exposure reduces proenkephalin (PENK) expression in the heart, aorta, and kidney and delays developing tolerance to morphine-induced locomotor hypoactivity:

A) PENK mRNA expression in female rats, B) PENK mRNA expression in male rats, C) distance traveled by female rats after 1 and 6 days of MOR injections, D) distance traveled by male rats after 1 and 6 days of MOR injections.

Data were analyzed by t-test for gene expression and paired t-test for locomotor activity and reported as mean ± SEM; n=6-8/group for gene expression and n=8 per group for locomotor activity test; * p<0.05 vs. control. VEH=vehicle, MOR=morphine, F=females, M=males, PENK=proenkephalin.

Prenatal MOR exposure sensitizes the locomotor response to morphine.

In adult rats, a single dose of morphine induced a depressant effect in locomotor activity that was similar between prenatal exposure to VEH and MOR (Fig. 6C). However, the development of tolerance after six injections of morphine was delayed in MOR-exposed compared to VEH-exposed counterparts (Fig. 6D). Data is presented pooling rats from both sexes since the disaggregated effect of MOR was similar in IUME offspring (Fig. S5).

Discussion

Our study is among the first to develop a rodent model to investigate the effects of prenatal morphine exposure on the offspring’s cardiovascular health outcomes from neonatal life to adulthood, shedding light on the potential link between prenatal MOR exposure and high blood pressure, a risk factor for the development of CVD. Although litter size and birth weight were similar between groups, IUME neonates were shorter and weighed less until weaning, showing a catch-up growth effect by the first month of life. As adults, IUME rats displayed increased sympathetic activation, blood pressure and blood and urine biomarkers associated with increased CVD risk. In addition, we identified endothelial dysfunction in MOR-exposed females as a potential sex-specific contributing risk factor.

OUD during pregnancy has been linked to low birth weight (LBW), preterm birth, birth defects, and NOWS ^2–5,18.Several factors associated with pregnancy outcomes have been shown to increase the risk of developing cardiovascular and metabolic diseases ^47–49. LBW and catch-up growth are established predictor of CVD risk, catch-up growth, a compensatory weight gain leading to rapid growth in infants or young children who are smaller for their age or adjusted age ⁵⁰. Causal factors include premature birth, perinatal malnourishment, or placental deficiency resulting in unhealthy growth patterns⁵¹. In addition, catch-up growth increases the risk of obesity, insulin resistance, aging, and non-communicable diseases ⁵².

Our study shows that IUME litters displayed a significantly reduced BW observed during lactation. As a limitation, this outcome does not allow determining whether the reduction in BW is secondary to prenatal programming or a result of a potential effect of opioids on milk production/composition and/or nursing-feeding behavior during lactation⁵³; consequently, it was not feasible to define the origin of the reduced BW during postnatal life. Thus, the design of studies with cross-fostering litters and milk composition assessment could provide insights into how IUME decreases BW and induces catch-up growth, in addition to studies designed to discern the effect of weigh loss during lactation from the opioids exposure on the cardiovascular phenotype. Also, IUME reduced tibia length through adulthood, suggesting the effect on body length is permanent; however, only IUME male rats showed reduced subcutaneous and visceral fat mass, indicating that the effect of IUME can impact the adipose tissue homeostasis mechanisms in a sex-specific manner. A potential explanation for the reduced fat mass could be given by in vitro studies in human and rodent adipocytes showing that EOPs promote lipolysis⁵⁴. However, the effect of prenatal opioids exposure on the metabolic programming of adipogenesis, adipocyte differentiation process and/or the sympathetic control of adipose tissue lipolytic function lipogenesis has not been systematically investigated. Therefore, one of the limitations of this study is the unfeasibility to define the origin of the reduced BW during postnatal life. Therefore, future studies will be needed to discern the effect of weight loss during lactation from opioid exposure on the cardiovascular phenotype.

OUD has been associated with the development of metabolic-like syndrome¹³. Tuduri et al. have shown that μ-OR stimulation increased insulin resistance and glucose intolerance, while a whole body μ-OR knockout improved glucose tolerance, insulin secretion, fatty acid oxidation, and insulin sensitivity⁵⁵, blocking α-ADR reduced sympathetic activation and metabolic dysfunction mediated by μ-OR⁵⁵. It also has been shown that acute and chronic opioid exposure are associated with alterations in lipid profile (increasing LDL, VLDL, aortic cholesterol content, and decreasing HDL), and islet cell damage in the pancreas^11,15. Accordingly, our data show that MOR-exposed offspring display a diabetic-like phenotype compatible with prolonged insulin resistance, evidenced by elevated FBG and reduced insulin levels. The contribution of a potential interaction between ORs and ADRs in liver, pancreas and/or adipose tissue from IUME offspring remains under investigation.

Our study showed that hypertension in IUME offspring is most likely from a neurogenic origin supported by reduced blood pressure in response to prazosin treatment and the greater depressor response to mecamylamine, along with the increased NE levels in plasma and urine. Nevertheless, IUME female rats displayed similar levels of NE compared with VEH. This lack in NE increase could be due to multiple factors. Females have reduced adreno-medullary derived synthesis of NE early in life, while after sexual maturity, estradiol can modulate the release, reuptake, and degradation. Therefore, although we found the expected reduced NE levels in females compared to males, IUME females may display mechanisms to enable a tight regulation of the NE metabolism despite increased sympathetic outflow. Studies in adult rats have shown that C1 adrenergic neurons in the rostroventrolateral medulla contain μ-ORs play a key role in blood pressure regulation during withdrawal. These neurons maintain tonic and reflex control of arterial pressure and sympathetic vasomotor tone.⁵⁹ In rat models, chronic administration of morphine is associated with μ-OR redistribution in C1 neurons,⁵⁸ while precipitated withdrawal induced by a μ-OR antagonist has been shown to reduce the availability of μ-OR in C1 neurons, increasing neuronal activation, which may facilitate the profound sympathetic hyperactivity observed in this model.

The EOS has been shown to interact centrally and peripherally with other GPCRs involved in the modulation of cardiovascular and sympathetic systems ^28,61,62. It has been shown that adrenergic and angiotensin receptors dimerize with μ-ORs in brain areas contributing to the pathogenesis of neurogenic hypertension by increasing the sympathetic tone. Further, EOPs can stimulate the release of catecholamines, increasing blood pressure, while α-ADR stimulation can increase the levels of β-endorphins^63,64. Spontaneously hypertensive rats (SHR) and other hypertensive animal models overexpress Gi proteins, the G proteins activated by ORs and α-ADRs, and are considered a contributing factor to the pathogenesis of hypertension^65,66. Furthermore, knocking out α-ADR eliminated the sympathoexitatory response to cocaine and morphine in mice⁶⁷, whereas blocking the μ-OR reduces plasma catecholamines and muscle sympathetic activation in humans⁶⁸. Further studies assessing tissue-specific α-ADRs and ORs expression and/or density, as well as their interaction, will clarify the possible mechanism by which IUME increases sympathetic activation and, consequently, blood pressure in our model. Interactions between ORs and ADRs can happen via ligand stimulation and/or dimerization, initiating a cascade of signaling increasing cardiovascular reactivity.

The heterodimerization of μ-OR can also occur with angiotensin type 1 receptor (AT1R), another GPCR implicated in blood pressure control^18,61,69. Numerous studies have investigated the interactions between the RAS and the sympathetic system^28,62,70,71. Centrally, EOPs, and morphine infusion inhibit AngII-induced pressor response, an effect that was reversed by naloxone administration. Peripherally, EOPs could indirectly mediate Ang II actions by increasing circulating levels of renin and angiotensin-converting enzyme (ACE). Additionally, ACE plays a role in endogenous opioid signaling by cleaving and degrading EOPs⁷². Furthermore, naloxone administration attenuated the development of hypertension in the SHR model known to show sympathetic and RAS activation^20,28,29. In the current study, the AngII-induced ex-vivo constriction was increased in IUME rats while the expression of AT1R and ACE were downregulated in the aorta,heart, and kidney (data not shown). This finding suggests the increased AngII-induced constriction is most likely facilitated by the ORs, potentially increasing the affinity to AT1R and amplifying the signaling cascade, as we showed that blocking ORs with naloxone can revert this effectherefore, IUME could induce chronic activation of the AT1R potentially enhanced by the heterodimerization with μ-OR, resulting in reduced AT1R expression. Notably, reduced levels of ACE in the vasculature could be linked to impaired degradation of enkephalins, leading to the co-stimulation of dimerized mu and AT1 receptors resulting in a potentiated constrictive effect. Despite these increases in AngII reactivity ex-vivo, IUME significantly attenuated the whole animal dose-response to acute doses of AngII only in female rats. While the isolated aorta is a conductance vessel, the acute pressor response primarily involves the contribution of several vascular beds of resistance vessels combined with the central effects of AngII combined with tissue-specific status of the EOS. Our study identified endothelial dysfunction in MOR-exposed female rats but not in males. Similarly, Cheon et al. reported that female mice showed a greater risk of morphine-induced vascular dysfunction, increased arterial stiffness, and resistance via the cofilin-ERK signaling pathway, suggesting that impaired endothelial function may contribute to the high blood pressure in female rats along with sympathetic activation⁷³. Taken together, the results from our study provide evidence that prenatal exposure to exogenous opioids may alter the programming and sensitivity of the vasculature to AngII in a sex-specific manner. Although the circulating levels of RAS components were similar between VEH and MOR-exposed rats, because of the discrepancy between in-vivo and ex-vivo responses, differences in RAS peptides at the central nervous system level cannot be ruled out.

Pre-clinical reports have shown opiates as important modulators of cardiovascular function in both normotensive and hypertensive states and studies have reported that EOPs are elevated in models of hypertension of various etiologies, including genetic and renovascular hypertension^29,74. The levels of EOPs fluctuate during the lifespan. Physiologically, β-endorphin plasma levels increase during pregnancy from week 6 (20 fmol/mL) to delivery (>120 fmol/mL) ⁷⁵. By PD5, EOPs levels in neonates and mothers decrease to physiological levels ⁷⁵. However, β-endorphin and met-enkephalin levels remain increased in opioid-exposed infants 40 days after birth⁷⁵, and continue to increase about 100-fold than young age-matched controls^75,76. Interestingly, pharmacologic treatments to reduce NOWS did not affect EOPs levels, and there was no relationship between NOWS symptom severity and EOPs plasma levels⁷⁷. ORs are expressed in the placenta and the brain of neonates in gestational weeks 12-13, where EOPs modulate brain development ^78,79. Therefore, prolonged exogenous stimulation of opioids could contribute to decreasing the energy level required for initiating an active cascade of GPCR signaling^76,80,81. These data support that exogenous prenatal opioid exposure could cause alterations in the EOS that have lifelong effects on cardiovascular and metabolic function. Therefore, fetal programming of the EOS could contribute to the phenotype depicted in our study and induce cardiovascular dysfunction and affect exogenous opioids response later in life, noting that specific periods of life and lengths of exposure affect the EOS status in a different manner.

In order to investigate the long-term behavioral consequences of IUME, we conducted a locomotor activity test in adult rats. Similar to a previous finding⁸², there was no difference in baseline locomotor activity between VEH and MOR prenatal exposed groups. In addition, a morphine dose (10mg/kg) known to produce acute hypoactivity ⁸² showed no difference in response between in-utero VEH and MOR groups. However, in response to repeated MOR exposure, healthy VEH rats became tolerant to the hypoactivity effect of MOR, whereas tolerance development was delayed in IUME rats. Thus, the neurobehavioral mechanism underlying tolerance to the behavioral effect of morphine appears to be impaired by IUME. While the mechanism underlying tolerance is not well understood, perhaps long-lasting alterations in opioid receptor densities in dopaminergic brain regions involved in locomotion may play a role⁸³.

Of note, this animal model does not rely on self-administration, an important component of OUD in humans; our study approach involved experimenter-delivered MOR. We used escalating doses to mimic the development of tolerance, one of the key features of OUD in humans, and the pain management regimens used in the clinical setting. Our primary goal was to model the effects of IUME on the offspring’s health outcomes, providing a valuable tool for testing potential interventions and therapeutic approaches, providing critical insight into the potential underlying mechanisms in the programming of hypertension in response to opioid exposure.

In conclusion, our study shows that in-utero opioid exposure affects blood pressure regulation and vascular function in adult female and male rats, increasing their future CVD risk. We showed that prenatal MOR exposure induces the dysregulation of EOPs expression in the heart, aorta, kidney, and liver and the sensitization to secondary MOR exposures and increased CVD biochemical risk factors. Understanding the molecular mechanisms by which morphine, a well-characterized opioid, impacts cardiovascular function will provide valuable fundamental knowledge to prevent and treat OUD-related cardiovascular comorbidities. In addition, this model will allow the investigation of mechanisms in opioids such as fentanyl, currently at the top of overdose death cases and challenging the conventional therapies to treat OUD and withdrawal syndrome in mothers and newborns.

Perspectives

This is the first study modeling the effect of in-utero opioid exposure on cardiovascular disease risk in female and male adult rats. Morphine exposure could induce changes in the EOS that may impact the regulation of cardiovascular function. This model will provide important insights for the mechanistic study of the effects of opioid exposure during developmental stages of life and pregnancy outcomes while establishing a valuable tool for testing interventions and potential therapeutic approaches for mothers and neonates.

What Is New?

• We established a pre-clinical model to investigate the long-term health effects of in-utero opioid exposure, wallowing us for the testing of potential interventions aiming to improve the pregnancy outcomes and the offspring’s health in addition to testing current therapies

• This study pinpoints dysregulation of the endogenous opioid peptides as a potential mechanism by which in-utero opioid exposure increases the cardiovascular disease risk in the adult offspring

• The identification of potential sex-specific risk to develop endothelial dysfunction and vascular reactivity that warrants the investigation of personalized approaches to reduce cardiovascular risk secondary to in-utero opioid exposure

What Is Relevant?

• It is challenging to predict the long-term impact on the cardiovascular health of children from women with opioid misuse, OUD, and opioid overdoses due to the lack of follow-ups later in life. This study provides insights regarding how opioids could affect the programming of the mechanisms regulating cardiovascular function.

• Investigating further the interaction of EOS with the mechanisms implicated in blood pressure regulation are required to understand potential targets for interventions.

• Understanding the effect of early-life opioid exposure on the endogenous opioid system programming may help identify potential biomarkers of risk factors for CVD. This study also highlights the importance of screening for in utero opioid exposure as a factor for increasing CVD risk.

Pathophysiological Implications?

• Prenatal morphine exposure affects fetal programming of the EOS and systems implicated in the regulation of cardiovascular and metabolic function

• Individuals with a history of prenatal opioid exposure can present comorbidities with sex-specific etiologies

• Along with the effect of morphine on the overall cardiovascular function, we identified a sex-specific dysregulation of the vascular function that involves mechanisms relevant to potential personalized medicine approach.

Summary

Opioids use during pregnancy profoundly affects the adult offspring’s cardiovascular function, activating the sympathetic system, increasing blood pressure, and impairing vascular function. The endogenous opioid system dysregulation secondary to the in-utero exposure to morphine could be mediating the overactivation of the principal central and peripheral systems in blood pressure regulation.

Nonstandard abbreviations:

MOR

Morphine

NOWS

Neonatal Opioid Withdrawal Syndrome

IUME

In Utero Morphine Exposure

OUD

Opioid Use Disorder

EOPs

Endogenous Opioid Peptides

EOS

Endogenous Opioid System

RAS

Renin Angiotensin System

BW

Body Weight