Vascular dysfunction in young, mid-aged and aged mice with latent cytomegalovirus infections.

Human cytomegalovirus (HCMV) is associated with vascular diseases in both immunosuppressed and immunocompetent individuals. CMV infections cycle between active and latent phases throughout life. We and others have shown vascular dysfunction during active mouse CMV (mCMV) infections. Few studies have examined changes in physiology during latent CMV infections, particularly vascular responses or whether the negative effects of aging on vascular function and fertility will be exacerbated under these conditions. We measured vascular responses in intact mesenteric and uterine arteries dissected from young, mid-aged, and aged latently mCMV-infected (mCMV genomes are present but infectious virus is undetectable) and age-matched uninfected mice using a pressure myograph. We tested responses to the α(1)-adrenergic agonist phenylephrine, the nitric oxide donor sodium nitroprusside, and the endothelium-dependent vasodilator methacholine. In young latently mCMV-infected mice, vasoconstriction was increased and vasodilation was decreased in mesenteric arteries, whereas both vasoconstriction and vasodilation were increased in uterine arteries compared with those in age-matched uninfected mice. In reproductively active mid-aged latently infected mice, mesenteric arteries showed little change, whereas uterine arteries showed greatly increased vasoconstriction. These vascular effects may have contributed to the decreased reproductive success observed in mid-aged latently mCMV-infected compared with age-matched uninfected mice (16.7 vs. 46.7%, respectively). In aged latently infected mice, vasodilation is increased in mesenteric and uterine arteries likely to compensate for increased vasoconstriction to mediators other than phenylephrine. The novel results of this study show that even when active mCMV infections become undetectable, vascular dysfunction continues and differs with age and artery origin.

Sphingosine 1-phosphate-induced vasoconstriction is elevated in mesenteric resistance arteries from aged female rats.

Sphingosine 1-phosphate (S1P), a bioactive lipid, signals through cell surface receptors to induce vasoconstriction and activate endothelial nitric oxide synthase (eNOS), suggesting a role for S1P in vascular tone modulation. Using a model of aging in female rats, we investigated the vasoactivity of S1P and the roles of eNOS and estrogen replacement in modulation of that vasoactivity. Mesenteric arteries from aged female rats were significantly more sensitive to S1P-induced vasoconstriction than arteries from young female rats, and reached greater maximum constriction (58.2+/-2.98 vs 34.8+/-4.44%; P<0.005). Modulation of this vasoconstriction by pretreating vessels with the NOS inhibitor l-NAME occurred only in young vessels. Ovariectomy reduced the maximum S1P-induced vasoconstriction observed in intact aged rats. Estrogen replacement did not appear to have an independent beneficial effect. However, estrogen replacement did restore nitric oxide modulation of S1P-induced vasoconstriction. Expression of the S1P(1) receptor, through which eNOS can be activated, was reduced in vessels from aged rats. S1P(1) receptor expression was restored in vessels from the estrogen-replaced group. S1P is a novel mediator of vascular tone through induction of both vasoconstriction and vasodilation. Reduced S1P(1) receptor expression on aging vessels may explain reduced eNOS activity, which results in greater sensitivity to S1P-induced vasoconstriction. Estrogen replacement in aging female rats restores both S1P(1) receptor expression and NOS activity, suggesting an important role for estrogen in this novel pathway of vascular tone modulation.

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles.

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.

Review: novel insights into the regulation of vascular tone by sphingosine 1-phosphate.

Endothelial dysfunction leading to increased vascular tone is implicated in the pathogenesis of cardiovascular disease, hypertension and pregnancy-related complications like preeclampsia and intrauterine growth restriction. Vascular tone is regulated by a balance between vasoconstrictor and vasodilator signals. Some vascular mediators circulate in blood, whereas others are produced by the endothelium and are delivered to the underlying vascular smooth muscle cells (VSMCs). It is proposed that increased permeability of resistance arteries in preeclampsia allows access of circulating vasoactive factors to VSMCs leading to increased vascular tone. This review focuses on the role of sphingosine 1-phosphate (S1P). This sphingolipid enhances the endothelial barrier, but it can also disrupt the barrier under certain conditions. These S1P-mediated effects on the endothelial barrier have been demonstrated in cultured endothelial cells and in isolated venules. They depend on S1P concentrations, the S1P receptors expressed and the vascular bed. However, no studies have examined if vascular tone is regulated by S1P in resistance arteries through changes in endothelial permeability and the leakage of circulating vasoconstrictors. Our recent studies using the pressure myograph system show that access of infused vasoconstrictors to VSMCs is blocked under low S1P concentrations. Pathophysiological levels of infused S1P disrupt the barrier and maximally increase vascular tone by facilitating access of itself and a co-infused vasoconstrictor to the VSMCs. Interestingly, infusion of an intermediate physiological concentration of S1P showed a small increase in endothelial permeability with controlled leakage of a co-infused vasoconstrictor that led to sub-maximal vascular tone development. These and other studies delineate the important role of S1P in the regulation of vascular tone and emphasize how dysfunction of this regulation can lead to pregnancy-related disorders.

IFPA Meeting 2009 workshops report.

Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.

Impaired vascular function in mice with an active cytomegalovirus infection.

Human cytomegalovirus (CMV) is implicated in vascular complications through endothelial dysfunction. However, the effect of in vivo infections on vascular function in isolated arteries has not been examined. In pregnancy, systemic and uterine vascular adaptations accommodate increased blood volume through several mechanisms, including decreased sensitivity to vasoconstrictors and increased production of endothelial-dependent vasodilators. We hypothesized that an active in vivo CMV infection would reduce vasodilation of isolated arteries to the endothelial-dependent vasodilator methacholine and increase vasoconstriction to the alpha(1)-adrenergic receptor agonist phenylephrine and that these CMV-induced changes would be accentuated in late pregnancy. A mouse CMV infection model was used to study vascular responses in isolated mesenteric and uterine arteries from nonpregnant and late pregnant mice. In the mouse, CMV is not transmitted to the fetus. Accordingly, there was no evidence of active infection in any fetus examined, even though an active infection was found in salivary glands, uterine and mesenteric arteries, and placentas. Contrary to our hypothesis, increased endothelial-dependent vasodilation was found in mesenteric arteries from infected compared with uninfected nonpregnant and pregnant mice These data implicate active CMV infections in hypotensive disorders. Similarly, increased vasodilation was found in uterine arteries from infected vs. uninfected nonpregnant mice. However, this was completely reversed in infected compared with uninfected late pregnant mice in which vasodilation in uterine arteries was significantly reduced. Uterine arteries from infected pregnant mice also showed increased vasoconstriction to phenylephrine. Maternal infection led to decreased placental weights but had no effect on fetal weights in late pregnancy. These novel data demonstrate abnormal systemic and uterine vascular responses during an active CMV infection in both nonpregnant and late pregnant mice. Importantly, despite reduced placental weights, fetal weights were maintained, suggesting effective intrauterine compensation in the mouse model.

Increased myogenic tone in 7-month-old adult male but not female offspring from rat dams exposed to hypoxia during pregnancy.

Intrauterine growth restriction (IUGR) increases the risk of cardiovascular disease later in life. Vascular dysfunction occurs in adult offspring from animal models of IUGR including maternal undernutrition, but the influence of reduced fetal oxygen supply on adult vascular function is unclear. Myogenic responses, essential for vascular tone regulation, have not been evaluated in these offspring. We hypothesized that 7-mo-old offspring from hypoxic (12% O(2); H) or nutrient-restricted (40% of control; NR) rat dams would show greater myogenic responses than their 4-mo-old littermates or control (C) offspring through impaired modulation by vasodilators. Growth restriction occurred in male H (P < 0.01), male NR (P < 0.01), and female NR (P < 0.02), but not female H, offspring. Myogenic responses in mesenteric arteries from males but not females were increased at 7 mo in H (P < 0.01) and NR (P < 0.05) vs. C offspring. There was less modulation of myogenic responses after inhibition of nitric oxide synthase (P < 0.05), prostaglandin H synthase (P < 0.005), or both enzymes (P < 0.001) in arteries from 7-mo male H vs. C offspring. Thus reduced vasodilator modulation may explain elevated myogenic responses in 7-mo male H offspring. In contrast, there was increased modulation of myogenic responses in arteries from 7-mo female H vs. C or NR offspring after inhibition of both enzymes (P < 0.05). Thus increased vasodilator modulation may maintain myogenic responses in female H offspring at control levels. In summary, vascular responses in adult offspring from adverse intrauterine environments are impaired in a gender-specific, age-dependent, and maternal insult-dependent manner, with males more profoundly affected.

Polarized release of human cytomegalovirus from placental trophoblasts.

Human cytomegalovirus (HCMV) is a ubiquitous infectious pathogen that, when transmitted to the fetus in utero, can result in numerous sequelae, including late-onset sensorineural damage. The villous trophoblast, the cellular barrier between maternal blood and fetal tissue in the human placenta, is infected by HCMV in vivo. Primary trophoblasts cultured on impermeable surfaces can be infected by HCMV, but release of progeny virus is delayed and minimal. It is not known whether these epithelial cells when fully polarized can release HCMV and, if so, if release is from the basal membrane surface toward the fetus. We therefore ask whether, and in which direction, progeny virus release occurs from HCMV-infected trophoblasts cultured on semipermeable (3.0-microm-pore-size) membranes that allow functional polarization. We show that infectious HCMV readily diffuses across cell-free 3.0-microm-pore-size membranes and that apical infection of confluent and multilayered trophoblasts cultured on these membranes reaches cells at the membrane surface. Using two different infection and culture protocols, we found that up to 20% of progeny virus is released but that <1% of released virus is detected in the basal culture chamber. These results suggest that very little, if any, HCMV is released from an infected villous trophoblast into the villous stroma where the virus could ultimately infect the fetus.

Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts.

Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.