Gestational Hypoxia and Developmental Plasticity.

Hypoxia is one of the most common and severe challenges to the maintenance of homeostasis. Oxygen sensing is a property of all tissues, and the response to hypoxia is multidimensional involving complicated intracellular networks concerned with the transduction of hypoxia-induced responses. Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. Hypoxia during gestation impacts both the mother and fetal development through interactions with an individual's genetic traits acquired over multiple generations by natural selection and changes in gene expression patterns by altering the epigenetic code. Changes in the epigenome determine "genomic plasticity," i.e., the ability of genes to be differentially expressed according to environmental cues. The genomic plasticity defined by epigenomic mechanisms including DNA methylation, histone modifications, and noncoding RNAs during development is the mechanistic substrate for phenotypic programming that determines physiological response and risk for healthy or deleterious outcomes. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue. The complex molecular and epigenetic interactions that may impact an individual's physiology and developmental programming of health and disease later in life are discussed.

Mitochondrial influences on smooth muscle phenotype.

Smooth muscle cells transition reversibly between contractile and noncontractile phenotypes in response to diverse influences, including many from mitochondria. Numerous molecules including myocardin, procontractile miRNAs, and the mitochondrial protein prohibitin-2 promote contractile differentiation; this is opposed by mitochondrial reactive oxygen species (mtROS), high lactate concentrations, and metabolic reprogramming induced by mitophagy and/or mitochondrial fission. A major pathway through which vascular pathologies such as oncogenic transformation, pulmonary hypertension, and atherosclerosis cause loss of vascular contractility is by enhancing mitophagy and mitochondrial fission with secondary effects on smooth muscle phenotype. Proproliferative miRNAs and the mitochondrial translocase TOMM40 also attenuate contractile differentiation. Hypoxia can initiate loss of contractility by enhancing mtROS and lactate production while simultaneously depressing mitochondrial respiration. Mitochondria can reduce cytosolic calcium by moving it across the inner mitochondrial membrane via the mitochondrial calcium uniporter, and then through mitochondria-associated membranes to and from calcium stores in the sarcoplasmic/endoplasmic reticulum. Through these effects on calcium, mitochondria can influence multiple calcium-sensitive nuclear transcription factors and genes, some of which govern smooth muscle phenotype, and possibly also the production of genomically encoded mitochondrial proteins and miRNAs (mitoMirs) that target the mitochondria. In turn, mitochondria also can influence nuclear transcription and mRNA processing through mitochondrial retrograde signaling, which is currently a topic of intensive investigation. Mitochondria also can signal to adjacent cells by contributing to the content of exosomes. Considering these and other mechanisms, it is becoming increasingly clear that mitochondria contribute significantly to the regulation of smooth muscle phenotype and differentiation.

Hypoxic modulation of fetal vascular MLCK abundance, localization, and function.

Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.

Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries.

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

Maternal Undernutrition Modulates Neonatal Rat Cerebrovascular Structure, Function, and Vulnerability to Mild Hypoxic-Ischemic Injury via Corticosteroid-Dependent and -Independent Mechanisms.

The present study explored the hypothesis that an adverse intrauterine environment caused by maternal undernutrition (MUN) acted through corticosteroid-dependent and -independent mechanisms to program lasting functional changes in the neonatal cerebrovasculature and vulnerability to mild hypoxic-ischemic (HI) injury. From day 10 of gestation until term, MUN and MUN-metyrapone (MUN-MET) group rats consumed a diet restricted to 50% of calories consumed by a pair-fed control; and on gestational day 11 through term, MUN-MET groups received drinking water containing MET (0.5 mg/mL), a corticosteroid synthesis inhibitor. P9/P10 pups underwent unilateral carotid ligation followed 24 h later by 1.5 h exposure to 8% oxygen (HI treatment). An ELISA quantified MUN-, MET-, and HI-induced changes in circulating levels of corticosterone. In P11/P12 pups, MUN programming promoted contractile differentiation in cerebrovascular smooth muscle as determined by confocal microscopy, modulated calcium-dependent contractility as revealed by cerebral artery myography, enhanced vasogenic edema formation as indicated by T2 MRI, and worsened neurobehavior MUN unmasked HI-induced improvements in open-field locomotion and in edema resolution, alterations in calcium-dependent contractility and promotion of contractile differentiation. Overall, MUN imposed multiple interdependent effects on cerebrovascular smooth muscle differentiation, contractility, edema formation, flow-metabolism coupling and neurobehavior through pathways that both required, and were independent of, gestational corticosteroids. In light of growing global patterns of food insecurity, the present study emphasizes that infants born from undernourished mothers may experience greater risk for developing neonatal cerebral edema and sensorimotor impairments possibly through programmed changes in neonatal cerebrovascular function.

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics.

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood-brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.

Prenatal metyrapone treatment modulates neonatal cerebrovascular structure, function, and vulnerability to mild hypoxic-ischemic injury.

This study explored the hypothesis that late gestational reduction of corticosteroids transforms the cerebrovasculature and modulates postnatal vulnerability to mild hypoxic-ischemic (HI) injury. Four groups of Sprague-Dawley neonates were studied: 1) Sham-Control, 2) Sham-MET, 3) HI-Control, and 4) HI-MET. Metyrapone (MET), a corticosteroid synthesis inhibitor, was administered via drinking water from gestational day 11 to term. In Shams, MET administration 1) decreased reactivity of the hypothalamic-pituitary-adrenal (HPA) axis to surgical trauma in postnatal day 9 (P9) pups by 37%, 2) promoted cerebrovascular contractile differentiation in middle cerebral arteries (MCAs), 3) decreased compliance ≤46% and increased depolarization-induced calcium mobilization in MCAs by 28%, 4) mildly increased hemispheric cerebral edema by 5%, decreased neuronal degeneration by 66%, and increased astroglial and microglial activation by 10- and 4-fold, respectively, and 5) increased righting reflex times by 29%. Regarding HI, metyrapone-induced fetal transformation 1) diminished reactivity of the HPA axis to HI-induced stress in P9/P10 pups, 2) enhanced HI-induced contractile dedifferentiation in MCAs, 3) lessened the effects of HI on MCA compliance and calcium mobilization, 4) decreased HI-induced neuronal injury but unmasked regional HI-induced depression of microglial activation, and 5) attenuated the negative effects of HI on open-field exploration but enhanced the detrimental effects of HI on negative geotaxis responses by 79%. Overall, corticosteroids during gestation appear essential for normal cerebrovascular development and glial quiescence but induce persistent changes that in neonates manifest beneficially as preservation of postischemic contractile differentiation but detrimentally as worsened ischemic cerebrovascular compliance, increased ischemic neuronal injury, and compromised neurobehavior.

MicroRNAs in brain development and cerebrovascular pathophysiology.

MicroRNAs (miRNAs) are a class of highly conserved non-coding RNAs with 21-25 nucleotides in length and play an important role in regulating gene expression at the posttranscriptional level via base-paring with complementary sequences of the 3'-untranslated region of the target gene mRNA, leading to either transcript degradation or translation inhibition. Brain-enriched miRNAs act as versatile regulators of brain development and function, including neural lineage and subtype determination, neurogenesis, synapse formation and plasticity, neural stem cell proliferation and differentiation, and responses to insults. Herein, we summarize the current knowledge regarding the role of miRNAs in brain development and cerebrovascular pathophysiology. We review recent progress of the miRNA-based mechanisms in neuronal and cerebrovascular development as well as their role in hypoxic-ischemic brain injury. These findings hold great promise, not just for deeper understanding of basic brain biology but also for building new therapeutic strategies for prevention and treatment of pathologies such as cerebral ischemia.

Long-term hypoxia uncouples Ca2+ and eNOS in bradykinin-mediated pulmonary arterial relaxation.

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ 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 Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) 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 Ca2+ with confocal microscopy. eNOS was inhibited with 100 µM NG-nitro-l-arginine methyl ester (l-NAME), PGI2 production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ 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 Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the ß1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

Chronic hypoxia alters fetal cerebrovascular responses to endothelin-1.

In utero hypoxia influences the structure and function of most fetal arteries, including those of the developing cerebral circulation. Whereas the signals that initiate this hypoxic remodeling remain uncertain, these appear to be distinct from the mechanisms that maintain the remodeled vascular state. The present study explores the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1), a potent vascular contractant and mitogen. In fetal lambs, chronic hypoxia (3,820-m altitude for the last 110 days of gestation) had no significant effect on plasma ET-1 levels or ETA receptor density in cerebral arteries but enhanced contractile responses to ET-1 in an ETA-dependent manner. In organ culture (24 h), 10 nM ET-1 increased medial thicknesses less in hypoxic than in normoxic arteries, and these increases were ablated by inhibition of PKC (chelerythrine) in both normoxic and hypoxic arteries but were attenuated by inhibition of CaMKII (KN93) and p38 (SB203580) in normoxic but not hypoxic arteries. As indicated by Ki-67 immunostaining, ET-1 increased medial thicknesses via hypertrophy. Measurements of colocalization between MLCK and SMαA revealed that organ culture with ET-1 also promoted contractile dedifferentiation in normoxic, but not hypoxic, arteries through mechanisms attenuated by inhibitors of PKC, CaMKII, and p38. These results support the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to ET-1 through pathways dependent upon PKC, CaMKII, and p38 that cause increased ET-1-mediated contractility, decreased ET-1-mediated smooth muscle hypertrophy, and a depressed ability of ET-1 to promote contractile dedifferentiation.

Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries.

Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca2+-sensitive K+ (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O2-5% CO2 in the presence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and ß1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle.NEW & NOTEWORTHY Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca2+-sensitive K+ (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets.

Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats.

BACKGROUND AND PURPOSE: Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH). METHODS: One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation. RESULTS: The marker proteins of vascular smooth muscle phenotypic transformation α-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of α-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits. CONCLUSIONS: rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth.

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca(2+)-activated K(+) (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca(2+). 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(2+) signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼ 140 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 >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca(2+) responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 µM nitro-L-arginine methyl ester or 10 µM 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 α1-subunit expression but increased ß1-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.

Vitamin D status and metabolism in an ovine pregnancy model: effect of long-term, high-altitude hypoxia.

Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy, and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α,25-(OH)2D in nonpregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α,25-(OH)2D compared with normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not increase further the plasma levels of 25-OH-D, but it significantly increased those of the active metabolite, 1α,25-(OH)2D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D-activating enzyme CYP27b1 and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung, with opposite effects on placental VDR. We conclude that ovine pregnancy is a model of increased vitamin D status, and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.

Chronic cerebrovascular dysfunction after traumatic brain injury.

Traumatic brain injuries (TBI) often involve vascular dysfunction that leads to long-term alterations in physiological and cognitive functions of the brain. Indeed, all the cells that form blood vessels and that are involved in maintaining their proper function can be altered by TBI. This Review focuses on the different types of cerebrovascular dysfunction that occur after TBI, including cerebral blood flow alterations, autoregulation impairments, subarachnoid hemorrhage, vasospasms, blood-brain barrier disruption, and edema formation. We also discuss the mechanisms that mediate these dysfunctions, focusing on the cellular components of cerebral blood vessels (endothelial cells, smooth muscle cells, astrocytes, pericytes, perivascular nerves) and their known and potential roles in the secondary injury cascade. © 2016 Wiley Periodicals, Inc.

Platelet-Derived Growth Factor Receptor-ß Regulates Vascular Smooth Muscle Cell Phenotypic Transformation and Neuroinflammation After Intracerebral Hemorrhage in Mice.

OBJECTIVE: Platelet-derived growth factor-BB activates platelet-derived growth factor receptor-ß and promotes vascular smooth muscle cell phenotypic transformation. Elevated levels of non-muscle myosin IIB (SMemb) are found in secretory smooth muscle cells along with inflammatory mediators, such as intercellular adhesion molecule-1, which can amplify neutrophil infiltration into the brain. In the present study, we investigated the role of platelet-derived growth factor-BB/platelet-derived growth factor receptor-ß following intracerebral hemorrhage-induced brain injury in mice, with emphasis on its ability to promote vascular smooth muscle cell phenotypic transformation followed by increased intercellular adhesion molecule-1 expression and elevated neutrophil infiltration in the vicinity of the hematoma. We also determined the extent to which plasmin from the hematoma influences the platelet-derived growth factor-BB/platelet-derived growth factor receptor-ß system subsequent to intracerebral hemorrhage. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: One hundred and fifty six eight-week-old male CD1 mice. INTERVENTIONS: Brain injury was induced by autologous arterial blood or plasmin injection into mouse brains. Small interfering RNA targeting platelet-derived growth factor receptor-ß was administered 24 hours before intracerebral hemorrhage. A platelet-derived growth factor receptor antagonist, Gleevec, was administered following intracerebral hemorrhage. A mitogen-activated protein kinase-activated protein kinase 2 inhibitor (KKKALNRQLGVAA) was delivered with platelet-derived growth factor-BB in naïve animals. Platelet-derived growth factor-BB was injected with a plasmin inhibitor (ε-aminocaproic acid) in intracerebral hemorrhage mice. Plasmin-injected mice were given platelet-derived growth factor receptor-ß small interfering RNA 24 hours before the operation. Neurological deficits, brain edema, western blots, and immunofluorescence were evaluated. MEASUREMENTS AND MAIN RESULTS: Platelet-derived growth factor receptor-ß small interfering RNA attenuated SMemb and intercellular adhesion molecule-1 expression and neutrophil infiltration at 24 hours post injury and reduced neurological deficits and brain edema at 24 and 72 hours following intracerebral hemorrhage. The platelet-derived growth factor receptor antagonist, Gleevec, reduced SMemb and intercellular adhesion molecule-1 expression. Platelet-derived growth factor receptor-ß activation led to increased expression of intercellular adhesion molecule-1 and was reversed by KKKALNRQLGVAA in naïve mice. Plasmin inhibition suppressed platelet-derived growth factor receptor-ß activation and neutrophil infiltration, whereas exogenous platelet-derived growth factor-BB increased platelet-derived growth factor receptor-ß activation, regardless of plasmin inhibition. Platelet-derived growth factor receptor-ß small interfering RNA decreased the expression of intercellular adhesion molecule-1 by plasmin injection. CONCLUSION: The platelet-derived growth factor-BB/platelet-derived growth factor receptor-ß system contributes to neuroinflammation through vascular smooth muscle cell phenotypic transformation near the hematoma via the p38 mitogen-activated protein kinase/mitogen-activated protein kinase-activated protein kinase 2 pathway following intracerebral hemorrhage. Plasmin is hypothesized to be upstream of the proposed neuroinflammatory system. The therapeutic intervention targeting the platelet-derived growth factor-BB/platelet-derived growth factor receptor-ß is a novel strategy to prevent plasmin-induced brain injury following intracerebral hemorrhage.

Imatinib attenuates cerebrovascular injury and phenotypic transformation after intracerebral hemorrhage in rats.

This study explored the hypothesis that intracerebral hemorrhage (ICH) promotes release of diffusible factors that can significantly influence the structure and function of cerebral arteries remote from the site of injury, through action on platelet-derived growth factor (PDGF) receptors. Four groups of adult male Sprague-Dawley rats were studied (n = 8 each): 1) sham; 2) sham + 60 mg/kg ip imatinib; 3) ICH (collagenase method); and 4) ICH + 60 mg/kg ip imatinib given 60 min after injury. At 24 h after injury, sham artery passive diameters (+3 mM EGTA) averaged 244 ± 7 µm (at 60 mmHg). ICH significantly increased passive diameters up to 6.4% and decreased compliance up to 42.5%. For both pressure- and potassium-induced contractions, ICH decreased calcium mobilization up to 26.2% and increased myofilament calcium sensitivity up to 48.4%. ICH reduced confocal colocalization of smooth muscle α-actin (αActin) with nonmuscle myosin heavy chain (MHC) and increased its colocalization with smooth muscle MHC, suggesting that ICH promoted contractile differentiation. ICH also enhanced colocalization of myosin light chain kinase (MLCK) with both αActin and regulatory 20-kDa myosin light chain. All effects of ICH on passive diameter, compliance, contractility, and contractile protein colocalization were significantly reduced or absent in arteries from animals treated with imatinib. These findings support the hypothesis that ICH promotes release into the cerebrospinal fluid of vasoactive factors that can diffuse to and promote activation of cerebrovascular PDGF receptors, thereby altering the structure, contractile protein organization, contractility, and smooth muscle phenotype of cerebral arteries remote from the site of hemorrhage.

miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids.

Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c.

Role of the sympathetic autonomic nervous system in hypoxic remodeling of the fetal cerebral vasculature.

Fetal hypoxia triggers compensatory angiogenesis and remodeling through mechanisms not fully elucidated. In response to hypoxia, hypoxia-inducible factor drives expression of cytokines that exert multiple effects on cerebral structures. Among these, the artery wall is composed of a heterogeneous cell mix and exhibits distinct patterns of cellular differentiation and reactivity. Governing these patterns are the vascular endothelium, smooth muscle (SM), adventitia, sympathetic perivascular nerves (SPN), and the parenchyma. Although an extensive literature details effects of nonneuronal factors on cerebral arteries, the trophic role of perivascular nerves remains unclear. Hypoxia increases sympathetic innervation with subsequent release of norepinephrine (NE), neuropeptide-Y (NPY), and adenosine triphosphate, which exert motor and trophic effects on cerebral arteries and influence dynamic transitions among SM phenotypes. Our data also suggest that the cerebrovasculature reacts very differently to hypoxia in fetuses and adults, and we hypothesize that these differences arise from age-related differences in arterial SM phenotype reactivity and proximity to trophic factors, particularly of neural origin. We provide an integration of recent literature focused on mechanisms by which SPN mediate hypoxic remodeling. Our recent findings suggest that trophic effects of SPN on cerebral arteries accelerate functional maturation through shifts in SM phenotype in an age-dependent manner.

Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of metyrapone.

Although the effects of prenatal undernutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering metyrapone, a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8 mo, offspring from Control, MFR, and MFR + Metyrapone groups were killed, and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal microscopy. Colocalization of smooth muscle α-actin (SMαA) with nonmuscle (NM), SM1 and SM2 myosin heavy-chain (MHC) isoforms was used to assess smooth muscle phenotype. Our results indicate that artery stiffness and wall thickness were increased, pressure-evoked myogenic reactivity was depressed, and myofilament Ca(2+) sensitivity was decreased in offspring of MFR compared with Control rats. MCA from MFR offspring exhibited a significantly greater SMαA/NM colocalization, suggesting that the smooth muscle cells had been altered toward a noncontractile phenotype. MET significantly reversed the effects of MFR on stiffness but not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2 colocalization. Together, our data suggest that MFR alters cerebrovascular contractility via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.