Vasotrophic Regulation of Age-Dependent Hypoxic Cerebrovascular Remodeling

Abstract

Hypoxia can induce functional and structural vascular remodeling by changing the expression of trophic factors to promote homeostasis. While most experimental approaches have been focused on functional remodeling, structural remodeling can reflect changes in the abundance and organization of vascular proteins that determine functional remodeling. Better understanding of age-dependent hypoxic macrovascular remodeling processes of the cerebral vasculature and its clinical implications require knowledge of the vasotrophic factors that influence arterial structure and function. Hypoxia can affect the expression of transcription factors, classical receptor tyrosine kinase factors, non-classical G-protein coupled factors, catecholamines, and purines. Hypoxia’s remodeling effects can be mediated by Hypoxia Inducible Factor (HIF) upregulation in most vascular beds, but alterations in the expression of growth factors can also be independent of HIF. PPARγ is another transcription factor involved in hypoxic remodeling. Expression of classical receptor tyrosine kinase ligands, including vascular endothelial growth factor, platelet derived growth factor, fibroblast growth factor and angiopoietins, can be altered by hypoxia which can act simultaneously to affect remodeling. Tyrosine kinase-independent factors, such as transforming growth factor, nitric oxide, endothelin, angiotensin II, catecholamines, and purines also participate in the remodeling process. This adaptation to hypoxic stress can fundamentally change with age, resulting in different responses between fetuses and adults. Overall, these mechanisms integrate to assure that blood flow and metabolic demand are closely matched in all vascular beds and emphasize the view that the vascular wall is a highly dynamic and heterogeneous tissue with multiple cell types undergoing regular phenotypic transformation.

INTRODUCTION

Vascular remodeling is crucial in maintaining homeostasis during development, exercise, and pregnancy. Blood vessels respond to their constantly changing environment by remodeling to match blood flow to local metabolic demand [1, 2]. Without proper regulation of perfusion, tissues can become ischemic and deprived of oxygen, resulting in cellular apoptosis, organ dysfunction, and eventually necrosis. Over the long term, the vasculature matches supply to demand by inducing capillary angiogenesis and by remodeling larger vessels upstream. A classic example of physiological remodeling is exercise conditioning, in which multiple factors induce long-term increases in maximum blood flow. To match the increased demand for oxygen and enable greater blood flow, existing large vessels undergo macrovascular remodeling while microvascular remodeling increases capillary density at the capillary level [3–6]. Capillary angiogenesis and collateral formation are examples of microvascular remodeling, a process distinctly different from macrovascular remodeling, in which structural changes occur within the walls of arteries and arterioles upstream from the capillaries. The ultimate example of macrovascular adaptation is pregnancy-induced remodeling of the uterine artery, a large conduit vessel that undergoes dramatic functional and structural changes throughout pregnancy [7]. Multiple types of microvascular and macrovascular remodeling are important not only in the mother but also in the developing fetus, especially during the transition from fetal to newborn life [8–10]. Given that the processes governing both microvascular and macrovascular remodeling remain poorly understood, particularly in the fetus and newborn, these processes warrant further research in fetal, newborn and adult arteries.

The principles governing homeostatic vascular remodeling also participate in pathophysiological remodeling in numerous diseases. For example, chronic hypertension can promote hypertrophic arterial remodeling through dynamic mechanisms [11]. Increased intraluminal pressures characteristic of chronic hypertension can alter vascular permeability, wall thickness, composition, and protein abundance [12]. Some of these changes are attributable to genetic factors that enhance inward arteriolar remodeling responses to increased luminal pressure [13] (Fig. 1). This type of remodeling of cerebral arteries can increase distensibility with reduced internal and external diameters [14] and thereby reduce the risk of aneurysms [15]. Other pathologies, such as subarachnoid hemorrhages, also induce cerebrovascular remodeling [16, 17] that is not compensatory but instead compromises flow-metabolism coupling and can even culminate in vasospasm. Clearly, both physiological and pathophysiological patterns of cerebrovascular remodeling are dynamic and regionally heterogeneous multifactorial processes influenced by the expression of numerous genes, receptors, and growth factors [18–20].

Fig. 1. Categories of Vascular Remodeling.

Remodeling can be hypertrophic, eutrophic, or hypotrophic. In hypertrophic remodeling, the medial cross-sectional area increases. In eutrophic remodeling, total medial cross-sectional area remains unchanged. In hypotrophic remodeling, cross-sectional area decreases. Remodeling that results in a reduction in luminal diameter with constant outside diameter is classified as inward remodeling (left panel). Remodeling that involves an increase in outside arterial diameter with a constant inside diameter is classified as outward remodeling (right panel). In eutrophic remodeling, both inside and outside diameters change. In the above panels, eutrophic remodeling is represented as a change in horizontal position with no vertical change. These combined structural changes profoundly influence the contractile characteristics of the individual smooth muscle cells within the medial layer.

To better understand the mechanisms governing vascular remodeling, it is important to differentiate functional from structural effects. Functional remodeling includes changes in vascular reactivity and contractility, which are fundamentally important for coupling of blood flow to local tissue metabolism. Such changes in function, however, are typically the consequence of changes in artery composition and structure. These structural changes can alter the abundance and organization of adventitial matrix proteins as well as the numbers and composition of individual cell types within the arterial wall [21–23]. Correspondingly, these alterations can increase outer arterial diameter (outward remodeling) or decrease luminal diameter (inward remodeling). In addition, total smooth muscle cell mass per unit length of artery can increase (hypertrophic remodeling) or remain unchanged (eutrophic remodeling) [11, 14, 24]. The extent to which these combined structural effects influence the contractile characteristics of the individual smooth muscle cells in the medial layer defines their functional consequences.

Smooth muscle cells are an integral component of the arterial wall and exhibit a phenotypic heterogeneity that is governed by local mechanical and chemical signals [21, 25, 26]. These cells can be classified as migratory, proliferative, synthetic, or contractile, and any single cell can exhibit mixtures of these and other characteristics [21]. Transitions among these phenotypes can be induced by either receptor tyrosine kinase (RTK) dependent growth factors [27, 28] or by non-classical G-protein coupled receptor (GPCR) ligands [29, 30]. In addition, smooth muscle cells can also undergo apoptosis, which is an important process in vascular remodeling [31]. Ultimately, the integrated effects of changes in the numbers, organization, and individual characteristics of the cellular components that make up the arterial wall determine the net result of remodeling.

Tissue hypoxia is a common feature shared among many types of both physiological and pathophysiological remodeling. This hypoxia drives remodeling to balance oxygen supply and demand at the cellular level through parallel microvascular and macrovascular effects. Most early studies of hypoxic remodeling focused on the pulmonary circulation, due largely to the clinical prevalence of persistent pulmonary hypertension of the newborn [32, 33]. These studies have established that mild chronic hypoxia directly increases pulmonary arterial pressure and promotes changes in vascular structure and function through coordinated actions of multiple vasotrophic factors [34]. Investigations of hypoxic vascular remodeling in other vascular beds are more rare and have focused predominantly on the functional consequences of varying durations of hypoxia, with emphasis on changes in vascular contractility and cardiac output distribution [35]. These effects are particularly prominent in the cerebral circulation, where a wide variety of studies have established that chronic hypoxia stimulate angiogenesis, increase capillary density, and reduce inter-capillary distances within the brain parenchyma [36–42]. The cerebral circulation is also subject to both functional and structural macrovascular remodeling, particularly in response to ischemic insults [43]. Virtually all of these remodeling responses are age-dependent and reflect the integrated action of a broad variety of both classical and non-classical vasotrophic factors [44].

As in the adult, fetal and newborn hypoxia can stimulate an increase in cerebral capillary angiogenesis and permeability [45, 46]. Chronic hypoxia can also compromise autoregulation and the dynamics of blood velocity in fetal and neonatal brains [47–49]. In addition, reactivity to nitric oxide (NO), a primary endogenous vasodilator released from the vascular endothelium, can be depressed by chronic hypoxia through reduced vascular soluble guanylate cyclase (sGC) activity [50]. These functional changes reflect underlying structural remodeling, including increased protein abundance and vascular smooth muscle proliferation in fetal arteries [23, 35, 51, 52]. Not surprisingly, the effects of hypoxia on both functional and structural remodeling vary considerably in fetal and adult arteries [53, 54]. The age-related differences are a consequence of the combined actions of multiple vasotrophic factors whose release and activity vary with age, vascular bed, and intensity of hypoxia.

The roles in vascular remodeling of known vascular growth factors and other non-classical vasotrophic factors remain uncertain, but have the potential to further understanding of vascular pathologies in both the fetal and adult cerebral circulations. To that end, it will be valuable to better appreciate how these factors function not just individually, but in combination in response to common pathophysiological stresses such as hypoxia. The present review therefore explores the main factors known to play a role in vascular remodeling, with emphasis on responses involving the fetal cerebral circulation where possible. Given the relative paucity of results directly related to the fetal cerebral circulation, the review is organized around the three main families of factors that govern overall vascular remodeling. The first of these are the transcription factors that have a global influence on vascular growth and differentiation.

TRANSCRIPTION FACTORS IN HYPOXIC VASCULAR REMODELING

An essential first step in the initiation of hypoxic vascular remodeling is the activation of pathways that can sense and respond to reduced oxygen availability. Low oxygen can function as a trigger, inducing downstream transcriptional and translational events that mechanistically regulate both microvascular and macrovascular remodeling. How hypoxia is detected and translated into changes in gene and protein expression was uncertain for many years prior to the discovery of Hypoxia Inducible Factor (HIF) by Semenza in 1992 [55]. The transcription factor HIF is now recognized as the main signal that activates cellular responses to hypoxia [56] (Fig. 2). It is a heterodimeric protein composed of α and β subunits, both of which are basic-helix-loop-helix (bHLH) proteins classified under the PAS family of transcriptional regulators [57]. Under normoxic conditions, HIF-1β is constitutively expressed whereas HIF-1α is continuously degraded via the ubiquitin-proteosome pathway [58]. Hypoxia inhibits prolyl hydroxylase, which is the oxygen-dependent enzyme governing HIF-1α ubiquitination and degradation [40]. Elevated levels of HIF-1α facilitate the formation of the HIF-1 complex, which then can bind to Hypoxia Responsive Elements (HRE) in the promoter regions of numerous genes and initiate transcription [56].

Fig. 2. Hypoxia and Transcription Factors.

Prolyl hydroxylase, the oxygen sensor, is responsible for HIF-1α ubiquitination and degradation under normoxic conditions. Hypoxia inhibits prolyl hydroxylase, leading to elevated levels of HIF-1. Accumulated HIF-1α can then facilitate the formation of the HIF-1 complex with constitutively expressed HIF-1β, which can then translocate to the nucleus where it binds to Hypoxia Responsive Elements (HRE) in the promoter regions of multiple genes and initiates transcription. Hypoxia-induced increases in TGF-β lead to Smad phosphorylation, which can also serve as coactivators for HIF-1α, and decrease PPARγ expression.

The effects of elevated HIF are highly heterogeneous among different tissues. This variability is due, at least in part, to tissue specific factors that influence HIF half-life and degradation. For example, products of HIF-sensitive genes can feedback through tyrosine kinase receptors or G-protein coupled receptors and regulate HIF levels [59, 60]. HIF-1α also can be upregulated by thrombin-α, PDGF-AB, and TGF-β1 in cultured and renal vascular smooth muscle cells [61]. Prostaglandin I2 (PGI2), a vasodilator with vasoprotective and antioxidant properties, can stabilize HIF-1α protein in hypoxic human umbilical vascular endothelial cells (HU-VECs). PGI2 appear to protect HIF-1α via inhibition of NADPH oxidase activity and reduction in levels of reactive oxygen species, which retard HIF-1α degradation [62]. HIF can also be regulated by Chloride Intracellular Channel 4 (CLIC4), which affects the upstream regulation and promotion of HIF and its downstream effectors, therefore influencing active transcription of HIF sensitive genes [63]. Studies of HIF turnover and half-life are a logical area for future research, particularly in situations where revascularization of transplanted tissues is essential for successful surgical outcomes [64, 65].

Another determinant of heterogeneity of local responses to HIF is the compliment of different active genes with HRE in their promoters, and the levels of transcription factors for the other cis-regulatory elements in each promoter region. For example, endothelial cells from various vascular beds differentially respond to hypoxia-induced HIF-1 by variably expressing endothelin-1, inducible nitric oxide synthase (iNOS), Fibulin-5, vascular endothelial growth factor-A (VEGF-A), VEGF receptors, and angiopoietin receptors [66–68]. In arterial smooth muscle, HIF can upregulate expression of low-density lipoprotein receptor-related protein [69]. Elevated levels of HIF-1α can also induce vascular remodeling under normoxic conditions in cultured vascular smooth muscle [70]. This range of effects emphasizes the versatility of diversity of HIF as a mediator of vascular responses to hypoxia.

In the brain, HIF can be used as a marker to identify hypoxia [71, 72]. Although upregulation of HIF-1α and its downstream effectors appear to be involved in vascular remodeling and hypoxic conditioning in both adult and neonatal brains [73–76], there have been no comparisons between adult and fetal HIF levels. Due to the fact that oxygen tensions are dramatically different between fetal and adult tissues, a logical speculation for future studies would be that HIF levels are adapted to the lower tissue oxygen tensions typical of the fetus. In the developing brain, HIF can directly influence proliferation of neuronal precursor cells [77]. HIF can also indirectly promote neuroprotection by stimulating expression of erythropoietin and VEGF [77–79]. Regionally, the effects of HIF are influenced by local conditions that determine whether HIF exerts either neuroprotective effects or neurotoxic effects through stimulation of apoptosis and necrosis [77]. Together, these results reflect the potential of HIF as a mediator of hypoxic vascular remodeling in the brains of both fetuses and adults.

The basic Helix-Loop-Helix structure of HIF-1α is also characteristic of Endothelial PAS protein 1 (EPAS1), a transcription factor closely related to HIF that might also contribute to the hypoxic remodeling response [80]. This transcription factor has had an interesting history owing to its independent discovery by at least three different research groups. Correspondingly, this factor has been named EPAS1 [81], HIF-1α-like factor (HLF) [82], and finally HIF-2α [83]. HIF-2α shares a 48% sequence identity with HIF-1α [84] and can be expressed during embryogenesis [85]. HIF-2α can induce cellular hypertrophy, reduce proliferation, and promote angiogenesis in neuroblastoma cells [86]. HIF-2α might also serve as a biomarker in advanced bladder cancer [87]. Interestingly, both HIF-2α and HIF-1α mRNA are distributed heterogeneously among all tissue and cell types [88] and can be expressed in the heart and lungs of neonates [81]. Both factors are stabilized by hypoxia and bind to HRE in multiple gene promoters [80]. As for HIF-1α, HIF-2α influences angiogenesis through upregulation of VEGF, and stimulates transcription of genes for EPO and the Tie-2 receptor [89–91]. During development, HIF-2α transcripts can be colocalized with HIF-1α transcripts, suggesting redundant roles that extend beyond embryogenesis that could include vascular stabilization and remodeling [92]. In relation to hypoxic remodeling, mutations of the EPAS-1 gene that codes for HIF-2α may have more beneficial effects for high altitude living than mutations of the EPO gene [93]. In addition, HIF-2α can inhibit ROS production by stimulation of antioxidant enzyme production [94]. Unlike HIF-1α, very little research has examined HIF-2α or its role in vascular development, maintenance or remodeling.

Another transcription factor involved in hypoxic remodeling is peroxisome proliferator-activated receptor gamma (PPARγ). Although traditionally associated with lipid metabolism and antioxidant protection during inflammation [95], it also plays a role in hypoxic vascular remodeling. Hypoxia stimulates an increase in TGF-β/Smad signaling that then downregulates PPARγ expression, functionally releasing a “brake” on remodeling [96, 97]. Hypoxic reductions in PPARγ thus promote remodeling and enable functional and structural changes to proceed. In the nucleus, PPARγ heterodimerizes with Retinoid X Receptor α (RXRα) and can then bind to peroxisome proliferator response elements (PPREs) on the promoter region of PPARγ target genes to induce transcription [98, 99]. In contrast to HIF, activation of PPARγ helps maintain vascular myogenic tone and attenuates remodeling [100, 101] by decreasing endothelial-derived ET-1 expression and inhibiting VEGF-induced angiogenesis [102]. PPARγ can also decrease VSMC proliferation and stimulate apoptosis [102].

In the cerebral vasculature, studies of PPARγ are rare but are attracting growing scientific interest. Cerebral arteries from mice with negative mutations in PPARγ exhibited reduced PPARγ levels and underwent both functional and structural remodeling [103]. Functionally, the arteries demonstrated impaired responses to agonist-induced vasodilation, which was attributed to elevated superoxide levels secondary to reduced antioxidant protection by PPARγ. Structural changes included increased distensibility, wall thickness, and cross-sectional area with decreased external diameter, as is typical of hypertrophic inward remodeling. Aside from the well-studied effects of PPARγ on lipid metabolism and inflammation, virtually nothing is known of the influence of hypoxia on PPARγ expression within the fetal cerebrovasculature, making this a promising topic for future investigation.

RECEPTOR TYROSINE KINASE-DEPENDENT VASOTROPHIC FACTORS

Whereas transcription factors exert effects only within the cells where they are synthesized, most growth factors are released into the extracellular space where they act as intercellular messengers. These messenger molecules, of which there are dozens, then activate cell surface receptors in either an autocrine or paracrine manner. One convenient method to classify these factors is according to the receptor type they bind and activate. For vasotrophic factors, the largest single class of receptors is the Receptor Tyrosine Kinase (RTK) family. In turn, the most widely studied vasotrophic factor that acts through RTK receptors is Vascular Endothelial Growth Factor [27].

Vascular Endothelial Growth Factor

VEGF was discovered more than six decades ago as the factor responsible for increased vascular permeability and was originally named Vascular Permeability Factor [104]. Subsequent studies identified VEGF as the main factor responsible for increased vascular permeability in tumors [105] and is now also recognized as the main vascular growth factor mediating angiogenesis [106, 107]. VEGF can also promote angiogenic effects, including tube formation, in cell cultures and can increase vascular endothelial cell proliferation in rat brains [108, 109]. On the other hand, under some conditions endothelial cells do not respond robustly to VEGF stimulation [110], suggesting that the role of the endothelium in remodeling is both heterogeneous and finely regulated. The VEGF family includes seven members (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF), which can act through three known receptor tyrosine kinases, VEGFR-1 (FLT-1), VEGFR-2 (KDR), and VEGFR-3 [111, 112]. Activation of these receptors can initiate highly variable and tissue type-dependent responses. For example, activation of VEGFR-2 can induce cell proliferation in endothelial cells [113], but can modulate contractile protein abundance in vascular smooth muscle [44]. In contrast, VEGFR-3 is expressed predominantly in lymphatic and venous vessels where it regulates lymphangiogenesis and sprouting [114]. Regulation of VEGF reactivity can function in an autocrine loop in which activation of either VEGF-R1 or VEGF-R2 can enhance mRNA and protein expression for VEGF-R1 in either its particulate or soluble form [115]. In turn, expression of VEGF-A, currently the most potent angiogenic protein known [112], can also be induced by TGF-β1 during tumor-induced angiogenesis [116].

A primary physiological stimulus for VEGF synthesis is hypoxia, which acts through HIF-1α to upregulate VEGF and other growth factors to promote homeostatic increases in capillary angiogenesis and vascular remodeling [117]. Hypoxia-induced HIF-1α can increase both VEGF-A and VEGFR-1 expression in endothelial cells derived from multiple different vascular beds [66] (Fig. 3). Hypoxic increases in VEGF within adjacent endothelial cells and pericytes can yield synergistic paracrine effects that enhance cellular growth and proliferation [118]. In some cell types, notably gliomas, hypoxia can also enhance VEGF levels through stabilization of VEGF mRNA [119, 120]. Not surprisingly, the effects of hypoxia on VEGF are highly tissue specific; VEGF levels can be unresponsive to hypoxia in the kidneys [121, 122].

Fig. 3. Effects of Hypoxia on Expression of Receptor Tyrosine Kinase-dependent Vasotrophic Factors.

Hypoxia-induced increases in HIF-1 levels can stimulate the transcription and translation of multiple Receptor Tyrosine Kinase-dependent vasotrophic factors. HIF-induced increases in FGF have been shown to stabilize HIF-1α, effectively enhancing its own synthesis. Increases in VEGF and VEGF receptors can induce endothelial cell proliferation. In addition to having angiogenic effects, VEGF can also be neuroprotective, can induce endothelial cell proliferation and vascular remodeling. VEGF can also activate PDGF receptors. Hypoxia causes an increase in VEGF, Angiopoietin 1 (Ang1), and PDGF-B levels, which activate Akt and inhibit apoptosis, particularly in neurons.

In the brain, the effects of hypoxia on VEGF have been widely studied owing to the potential of VEGF to facilitate recovery from ischemic cerebral insults [123]. These benefits are due not only to the ability of VEGF to stimulate cerebral angiogenesis [40, 41, 124], but are due also to VEGF’s neuroprotective properties in both mature [79, 125] and immature brain [126]. An important component of this overall effect is that hypoxia upregulates expression of VEGF mRNA and protein in the brain [107, 127, 128]. VEGF also can enhance its efficacy in the brain through upregulation of VEGFR-2 mRNA [129]. All cerebral cell types appear to participate in this pattern of responses, including astrocytes, which exhibit increased expression of VEGF following hypoxic exposure [130]. Interestingly, the cellular sources of VEGF are highly age dependent such that VEGF is expressed primarily in neurons of the immature brain, but in both neurons and glial cells of the mature brain [124]. Aside from these differences, sustained hypoxia increases VEGF expression in both neurons and glia, regardless of postnatal age. Based on studies in large arteries [44], these hypoxic increases in VEGF could potentially contribute to hypoxic cerebrovascular remodeling in an age-dependent manner. This hypothesis awaits future experimental confirmation.

Platelet Derived Growth Factor

Platelet-Derived Growth Factor (PDGF) has long been recognized as a major influence on vascular growth and development, particularly in developing tissues [131, 132]. It is a dimeric polypeptide with extensive homology to the peptide sequences of VEGF [133]. One major consequence of this homology is that receptors for PDGF can be activated not only by PDGF, but by VEGF as well [134, 135]. Active PDGF ligands can be composed of any pair of four different isoforms, designated as A, B, C, and D. The most common pairs, biologically, are PDGF-AA, PDGF-AB, and PDGF-BB [136] and thus the A and B forms have been most widely studied. Polypeptides, A and B, are transcribed from different genes but can be dimerized by a disulfide bond [137, 138]. The receptors that bind active PDGF dimers are composed of two different subunits, an α-subunit (PDGFR-α), which can bind both A and B chains, and a β-subunit (PDGFR-β), which can bind B-chain only. These subunits can associate reversibly to bind specific PDGF ligands [139]. Most importantly, different PDGF ligands produce different cellular responses even when acting on a common receptor [140]. PDGF can stimulate mitogenesis in smooth muscle, NO-dependent vasorelaxation in endothelium-intact aortic rings [141], and microvascular angiogenesis in invasive breast cancer [142]. PDGF-BB can transform smooth muscle to a less contractile phenotype, and is crucial for proper lung development of neonatal rats [28, 143].

As for most vasotrophic factors, the levels of PDGF and its receptors in any tissue are subject to regulation by many different influences. PDGFR-α levels can be upregulated by basic fibroblast growth factor (FGF-2), which can facilitate smooth muscle proliferation upon subsequent stimulation with PDGF-AA [144]. Alpha-thrombin can also increase mRNA levels for PDGF-A and simultaneously decrease mRNA for PDGFR-β in smooth muscle [145]. Hypoxia is also an important modulator of PDGF signaling in many different tissues. Although hypoxia has little effect on renal expression of PDGF-A and PDGF-B [121, 122], hypoxia can markedly increase transcription of the PDGF-B gene in HU-VEC cultures [138]. In rat lung parenchyma, hypoxia can transiently increase PDGF-B mRNA levels [146]. In neonatal rat lung, hypoxia increased mRNA for PDGF-B, PDGFR-α and PDGFR-β but decreased the apparent protein abundance of PDGF-A, PDGF-B and PDGFR-α [143], suggesting important hypoxic effects on the stability and translation efficiency of these mRNAs. In pulmonary arterial smooth muscle of neonatal rats, chronic hypoxia increased proliferation and expression of both PDGF-BB and PDGFR-β [147]. Hypoxia also appears to mediate PDGF-dependent hyperphosphorylation of PDGFR-β, and thereby enhance pulmonary artery endothelial and smooth muscle proliferation [148, 149].

Within the central nervous system, PDGF is crucial for recruitment of pericytes involved in brain capillary angiogenesis during embryonic development [136, 150]. Recruited pericytes can produce other vasotrophic factors such as TGF-β and VEGF, and are crucial in initiation, guidance, extension, and maturation of vessels [117]. In areas of focal ischemic cerebral infarct, injured tissue expresses increased levels of mRNA and protein for both PDGF-B and PDGFR-β [136]. More directly, hypoxia can increase mRNA and protein levels for PDGF-B in human glioblastoma cells [151]. In neurons, hypoxia can also increase mRNA and protein for PDGF-B and subsequent phosphorylation of PDGFR-β, leading to Akt activation and attenuation of apoptosis [152]. Effects of hypoxia in the central nervous system also appear to be regionally heterogeneous; chronic hypoxia can depress the abundance of PDGFR-β receptors in the dorsocaudal brainstem and simultaneously increase mRNA levels for PDGF-B and PDGFR-β in the solitary tract nucleus [153–155]. Together, these results demonstrate that, as for VEGF, the effects of hypoxia on PDGF signaling are highly dependent on age and cell type. Similarly, the roles of PDGF in hypoxic cerebrovascular remodeling remain largely unexplored, particularly in the immature brain.

Angiopoietins

Many of the vascular effects of hypoxia are attributable to the factors whose expression is upregulated by the actions of HIF-1α. In addition to VEGF, HIF-1α also drives the expression of angiopoietins, growth factors crucial for vascular maintainance and induction of vessel sprouting [156]. HIF increases angiopoietin-2 (Ang2) levels via a COX-2 dependent increase of prostaglandin E2 [157]. Four types of angio-poietin have been identified, including Ang1, Ang2, Ang3, and Ang4, all of which play a role in vascular and lymphatic remodeling in the adult mice [158, 159]. In endothelial cells, however, Ang1 and Ang3 exhibit few mitogenic effects [160, 161]. Expression of angiopoietins in vascular cell types is also heterogeneous; vascular smooth muscle expresses both Ang1 and Ang2 but endothelial cells primarily express just Ang2 [162]. As for VEGF, angiopoietins can also be anti-apoptotic, particularly in endothelia and mesenchymal stem cells [163, 164]. The receptors for angiopoietins are members of the RTK family and include Tie1 and Tie2 [162, 165]. In combination with these receptors, Ang1 and Ang2 operate in a push-pull manner in which Ang2 destabilizes, and Ang1 stabilizes, vessels undergoing angiogenesis [166–168]. To achieve this effect, Ang2 inhibits binding of Ang1 to Tie2 and thereby destabilizes capillaries and helps initiate microvascular angiogenesis. In addition, Tie1 also reciprocally regulates the binding of Ang1 and Ang2 to the Tie2 receptor to control responses to angiopoietin stimulation [169].

In relation to vascular remodeling, angiopoietins act in concert with VEGF [114]. Together with VEGF, Ang1 promotes increased arterial lumen diameter and Ang2 acts to extend vessel length and increase propagation of sprouting cells [117, 168]. Both VEGF and FGF-2 can increase Ang2 in microvascular endothelial cells, which can antagonize the effects of Ang1 and promote disassembly of the vascular wall and formation of new vessel sprouts [170]. Conversely, TGF-β1 can decrease Ang2 production. Ang1 and Ang2 can also decrease Ang2 production through negative feedback at the mRNA level. Correspondingly, expression of angio-poietin receptors are also subject to physiological regulation through which FGF-2 and VEGF, either alone or in combination, can increase Tie1 expression. Similarly, Tie2 expression can be increased by FGF-2, Ang1, or Ang2 [162]. Clearly, the angiopoietins are another category of important vasotrophic factors whose complex influences are governed by the simultaneous actions of multiple physiological influences.

One key determinant of angiopoietin actions is hypoxia. Hypoxia can upregulate Ang2 mRNA and protein levels in all major categories of cells [171–173]. In endothelial cells, hypoxia-induced increases in HIF-1 produce reciprocal increases in Ang2 and Tie2 expression but decreases in Ang1 expression [66, 173, 174]. Hypoxia also can increase both the transcription and stability of Ang2 mRNA in HUVECs [157]. Hypoxia can regulate Ang2 expression indirectly, at least in HUVEC cultures, through HIF-induced increases in COX-2 and subsequent increases in prostacyclin and prostaglandin E2, which in turn can increase Ang2 levels under either normoxic or hypoxic conditions [157].

Within the central nervous system, angiopoietins and their receptors can be expressed by neurons as well as by cerebrovascular cell types. Ang1 promotes Akt phosphorylation in neurons, and thereby inhibits caspase-3 activation and attenuates apoptosis [175]. In cerebrovascular endothelial cells, hypoxia and ischemia can increase Ang2 mRNA and protein without effects on Ang1 or Tie2 [176, 177]. Cerebral ischemia also can promote transient and region specific changes in Tie1 and Tie2 expression that correspond with regional changes in cerebral blood flow [178]. Most interestingly, regions exhibiting increased angiogenic activity also demonstrated colocalization among Tie2, Ang2, FGF-2 and VEGF, emphasizing the critical role of interactions among factors involved in vascular remodeling [178]. To date, most studies of the roles of angiopoietins in cerebrovascular remodeling have focused on their contribution to responses of the cerebral microcirculation to ischemia [179]; systematic assessments of the effects of hypoxia alone on participation of angiopoietins in cerebrovascular remodeling have yet to be performed. Such studies could be particularly illuminating in regards to control of physiological cerebral angiogenesis and remodeling, particularly in the immature cerebral circulation where low oxygen tension and high prostaglandin concentrations are typical.

Fibroblast Growth Factor

The fibroblast growth factor (FGF) family includes 22 members that can act on any of the four FGF tyrosine kinase receptors [180]. As established mitogens for endothelial cells, basic fibroblast growth factors (FGF-2) can initiate angiogenesis by inducing endothelial cell proliferation and cord formation [181]. As for other angiogenic growth factors, FGFs are synergistic with VEGF and other vasotrophic factors in their ability to promote capillary formation [167]. The production of FGF-2 by capillary endothelial cells can act in an autocrine manner to stimulate further endothelial cell proliferation [182]. In addition to these autocrine effects, FGF-2 can regulate expression of other factors. For example, FGF-2 can upregulate PDGFR-α levels, allowing smooth muscle cells to become more responsive to PDGF-AA stimulation [144]. FGF-2 itself is subject to upregulation by PDGF-BB and TGF-β in VSMCs [183]. In relation to vascular remodeling, a particularly important effect of FGF-2 is its ability to induce morphological, and possibly phenotypic, transformation in aortic smooth muscle [184]. Such effects may be particularly important during hypoxia, in which FGF-2 can increase ROS production, stabilize HIF-1α and other ROS-sensitive transcription factors, and increase its own transcription and translation in an autocrine manner [148, 185, 186]. During episodes of postnatal chronic hypoxia, FGF-2 levels can be increased heterogeneously among different brain regions and are particularly prominent in immature glial cells [187]. In hypoxic neurons, FGF-2 may also improve neuronal survival, contribute to hypoxic conditioning and serve a neuroprotective role [186, 188, 189]. These neuroprotective effects can be observed also in hypoxic-ischemic neonatal rat brain [190]. Interestingly, FGF-2 appears to increase proliferation, retard maturation, and hinder differentiation of neural progenitor cells [191]. How FGF-2 affects vascular smooth muscle progenitor cells is unknown. This raises the untested possibility that a portion of the neuroprotective effects of FGF-2 following an interval of hypoxia may be attributable to potential protective effects on the multiple cell types that make up the arterial wall.

RECEPTOR TYROSINE KINASE-INDEPENDENT VASOTROPHIC FACTORS

The ability of hypoxia to promote vascular remodeling is clearly a consequence of a highly dynamic interplay among multiple vasotrophic factors and physiological influences. As indicated above, growth factors dependent upon tyrosine kinase receptors constitute a major component of this regulation. However, the vasotrophic factors involved in hypoxic remodeling also include many other growth factors that act independently of RTKs. One of the best studied of these RTK-independent vasotrophic factors in Transforming Growth Factor β.

Transforming Growth Factor β

The transforming growth factor beta (TGF-β) superfamily consists of three main isoforms, TGF-β1, TGF-β2, and TGF-β3 [192, 193], all of which can promote angiogenesis or vessel regression in tumors [194, 195]. TGF-β1 can decrease endothelial tube formation and cause capillary-like structures to regress [167]. The receptors for TGF-β molecules are serine-threonine kinases that phosphorylate Smad proteins, leading to their translocation to the nucleus where they alter transcription of numerous genes [196]. In smooth muscle cells, TGF-β1 can promote differentiation but is only one of many factors governing this process [197]. Of particular importance for vascular remodeling are the antagonistic interactions between TGF-β1 and FGF-2. In this context, either decreased FGF-2 or increased TGF-β1 can induce pericyte differentiation and expression of α-smooth muscle actin, leading to differentiation of the contractile smooth muscle phenotype [198].

Hypoxia can increase TGF-β2 mRNA and protein levels and enhance Smad2 and Smad3 phosphorylation in endothelial cells [199] (Fig. 4). Hypoxia-induced HIF-1 also binds Smad proteins, which serve as coactivators and thereby contribute to hypoxic vascular remodeling [200, 201]. Increases in TGF-β expression can induce G protein–coupled receptor kinase 2 (GRK2), a downstream effector of TGF-β, to desensitize G-protein coupled receptors via negative feedback, terminate TGF-β/Smad signaling, and inhibit Ang2-induced proliferation [202]. In the brain, TGF-β1 secreted by microglia and macrophages contribute to cerebrovascular remodeling following a focal ischemic insult [203]. These effects, together with the ability of TGF-β1 to inhibit microglial activation, help explain why TGF-β1 can be neuroprotective following hypoxic-ischemic insults [204–208]. Despite these many effects of TGF-β on vascular development and differentiation, systematic studies of the roles of this growth factor in normal growth and development of the cerebral vasculature have yet to be undertaken, particularly in relation to the vascular effects of hypoxia.

Fig. 4. Hypoxia has heterogeneous effects on Receptor Tyrosine Kinase-independent vasotrophic factor signaling across various cell types.

In perivascular nerves, hypoxia inhibits the synthesis and decreases the content of NE while enhancing serotonin (5-HT) synthesis. Elevated 5-HT levels can then induce proliferation of smooth muscle cells and increase prostacyclin levels, which inhibits ROS production and increase Ang2 production. Adenosine can activate A₂ receptors and inhibit proliferation while ATP enhances mitogenesis in SMCs but can also increase endothelial cell proliferation. Hypoxia enhances the expression of TGF-β, preproET-1 and ET-1 while inhibiting NO synthesis in endothelial cells. Hypoxia also enhances expression of both ET receptors in smooth muscle, thereby enhancing the effects of ET-1. Increased TGF-β2 levels enhance Smad2/Smad3 phosphorylation, which can then act as a coactivator for HIF-1. Angiotensin II activates AT₁, which leads to an increase in FGF-2, PDGF, TGF-β, and NADPH oxidase. Increased NADPH oxidase leads to enhanced ROS production, which can inhibit NO bioavailability and induce hypertrophy and hyperplasia of smooth muscle cells. ROS can also increase the gene expression of HIF-1α and stabilize the HIF-1α protein. The HIF-1 complex then enters the nucleus, binds HREs, and results in increased transcription of VEGF, VEGF receptors, and Ang II. The diagram includes separate depictions of mechanisms in neurons, smooth muscle cells, and endothelial cells. For reference, a generic (parenchymal) cell is depicted in the lower left corner.

Nitric Oxide

The vascular endothelium plays a critical role in regulating active vascular tone under both normoxic and hypoxic conditions through the release of two main vasoactive factors, nitric oxide (NO) and endothelin (ET), with generally opposing effects on contractility [209, 210]. In addition to their well-documented vasomotor roles, however, both of these factors also exert continuous and opposing trophic influences on adjacent smooth muscle. Given that hypoxia increases endothelin synthesis but decreases NO synthesis and release, both of these factors are important contributors to hypoxic vascular remodeling [66, 211].

The vasorelaxant characteristics of nitric oxide arise largely from its ability to activate soluble guanylate cyclase and increase cGMP synthesis, which activates the serine-threonine kinase Protein Kinase G (PKG) [210, 212, 213]. PKG, in turn, can phosphorylate a broad variety of substrates within smooth muscle, including transcription factors such as CREB that govern smooth muscle phenotype [214, 215]. Aside from smooth muscle, PKG can also play a role in endothelial cell differentiation and tube formation [216]. Apart from PKG, NO can also downregulate expression of other vasotrophic factors, including preproET-1 and PDGF-B [217].

Physiological release of endothelial nitric oxide is governed primarily by fluid shear stress. Not only does shear stress promote the immediate release of NO, it also can upregulate eNOS mRNA and the long-term capacity for NO release [218]. Levels of eNOS are also increased by FGF-2 [216]. Similarly, stimulation of the insulin receptor can activate PI3K and Akt pathways to induce NO production, suggesting that changes in insulin receptor density influence NO release [219]. Through activation of the ETA receptor, endothelin can also upregulate expression of eNOS in pulmonary vascular endothelium [220]. Because oxygen radicals can rapidly inactivate NO [107], any long-term change in anti-oxidant activity also changes NO action on adjacent smooth muscle. Statin treatment can also increase NO bioavailability in fetal sheep, most probably through an increased capacity for NO synthesis [221]. Equally important, the capacity for NO synthesis and release in most vascular beds increases with developmental age [222, 223], which helps explain certain age-related differences in reactivity to endothelium-dependent vasodilators [224] but also predicts that the role of NO in vascular remodeling strengthens with advancing postnatal age.

Under conditions of hypoxia, changes in NO production are highly heterogeneous and depend on the duration and intensity of hypoxia in an organ specific manner. In the hypoxic lung, NO can promote angiogenesis and ameliorate hypoxic pulmonary hypertension [225]. Hypoxia also increases eNOS mRNA in the pulmonary vasculature, which helps attenuate pulmonary remodeling and hypertrophy [220, 226]. In contrast, in the cerebral and femoral vasculatures, NO production is depressed, which compromises NO-dependent stabilization of contractility and promotes remodeling [227, 228]. Attenuation of the capacity for NO release by chronic hypoxia is further enhanced by simultaneous reductions in sGC activity in both fetal and adult arteries [50]. In parallel, chronic hypoxia enhanced neuronal NOS expression in fetal brain homogenates [229] but depressed nNOS expression in the perivascular nerves innervating middle cerebral arteries [230], suggesting that hypoxia exerts opposite and tissue specific effects on NO production within the brain. Most interestingly, cerebral expression of eNOS, nNOS and iNOS were all increased following recovery from hypoxia, demonstrating that overall regulation of NO production is very tightly controlled. Altogether, these findings emphasize that NO stabilizes the contractile phenotype but is only one of many factors that contribute to the highly integrated, multifactorial processes determining vascular differentiation and remodeling, particularly during sustained hypoxia.

Endothelins

The discovery that vascular endothelium mediates acetylcholine-induced vasodilatation [231] through the release of NO [232] motivated numerous follow-up studies of other possible endothelium-derived vasoactive factors. In 1988, Yanagisawa reported that in addition to NO, the endothelium also releases endothelin (ET), one of the most potent endogenous vasoconstrictors ever discovered [233]. Three isoforms of endothelin have been identified (ET-1, ET-2, and ET-3) and these activate two separate endothelin receptors (ETA and ETB) [234]. The two ET receptors display distinct affinities for each ET subtype and often exhibit opposing actions; ETA can induce vasoconstriction whereas ETB can stimulate vasodilation, depending on the location and distribution of each receptor type [235, 236]. In some situations, ET can also induce release of vasodilators [237, 238].

Endothelin is implicated in many diseases, especially in hypertension-induced remodeling [239]. ET appears involved in hypertension-induced hypertrophy of cerebral arteries without changing their distensibility [240]. Diabetic mice can also display increased ET receptor levels and ET-1 dependent matrix metalloproteinase activation, which can facilitate cerebrovascular remodeling, especially after hypoxic exposure [241, 242]. Through binding to ETA receptor, increased ET levels can activate the transcription factor Nuclear Factor of Activated T cells, isoform 3 (NFAT3), resulting in hypertension and vascular remodeling. In smooth muscle, NFAT3 can increase smooth muscle α-actin mRNA and contribute to increased cross-sectional wall thickness in mesenteric arteries [243].

Expression and release of ETs are regulated by a broad variety of influences. Importantly, ETs can be produced by non-endothelial cell types, including vascular smooth muscle, although at a much lower rate than by endothelial cells [244]. Levels of ET mRNA in cultured human vascular smooth muscle can be enhanced by numerous vasotrophic factors including Ang2, TGF-β, and PDGF-AA [245]. In pulmonary artery smooth muscle, TGF-β can directly enhance expression of mRNA for preproET-1 (ET-1 precursor) and thereby increase ET-1 expression [246, 247]. In feedback fashion, hypoxia-induced increases in RTK-dependent growth factors (FGF-1, FGF-2, and PDGF-BB), but not G-protein coupled vasotrophic factors (Angiotensin-II and ET-1) can upregulate ETA expression in cultured pulmonary artery smooth muscle [248].

Hypoxia is a particularly important regulator of ET expression in many vascular beds. In the rat kidney, hypoxia increases ET-1 expression [121]. In the rat pulmonary circulation, hypoxia can increase both pulmonary and plasma ET expression [249]. In mouse and human pulmonary artery endothelial cells, hypoxia can increase expression of not only ET-1, but also Endothelin Converting Enzyme-1, ETA, and ETB [250]. Chronic hypoxia can also increase mRNA levels for preproET-1 and ET-1 protein in pulmonary smooth muscle and epithelium together with increased medial thickness of bronchiolar arteries [251]. In turn, hypoxic increases in ET-1 can be attenuated by PPARγ activation [250]. Interactions between NO and ET, both of which are modulated by hypoxia, also affect the hypoxic remodeling response. In this manner, endothelium derived NO can attenuate hypoxic remodeling and medial hypertrophy secondary to increased ET-1 levels [226]. NO can also downregulate ET-1 levels and this effect can be strong enough to abrogate hypoxia-induced increases in ET-1 mRNA and protein in endothelial cells [217]. In feedback fashion, hypoxic increases in ET-1 can act through the ETA and ETB receptors to elevate eNOS mRNA in the pulmonary vasculature while also increasing circulating hematocrit and ET-1 levels. These increased ET-1 levels promote thickening of the medial layer in pulmonary arteries [220].

In the normoxic central nervous system, neurons and endothelial cells express preproET-1, and neurons also express both ETA and ETB receptors [252, 253]. Following a hypoxic-ischemic insult, ET-1 expression is upregulated primarily in endothelial and glial cells [253, 254]. Hypoxia-ischemia can also upregulate ETA and ETB receptors in cerebral arteries [255]. Such changes in ET-1 signaling pathways can have important consequences for post-ischemic recovery, given that ET-1 can reduce cerebral perfusion under normoxic, hypoxic, and hypercapnic conditions, such as those typical of the post-ischemic brain [256]. Consistent with this possibility, overexpression of ET-1 can compromise blood-brain-barrier integrity and enhance edema following an ischemic cerebral insult [257]. In addition, by virtue of its properties as an endogenous ET antagonist [258], the hormone relaxin has the potential to ameliorate ET-induced cerebrovascular remodeling [259]. This hypothesis awaits direct experimental confirmation, as does the more general hypothesis that age-dependent hypoxic cerebrovascular remodeling is mediated, at least in part, by increased ET-1 effects on cerebral arteries.

Angiotensin II

The renin-angiotensin system is best known for its critical roles in regulation of salt and water balance, and how dysfunction of this regulation can lead to hypertension. Hypertension associated with elevated production of angiotension, in turn, can also lead to secondary changes in vascular structure and function [260, 261]. Some of this remodeling, however, may be due to direct vasotrophic effects of angiotensin II on vascular smooth muscle [262, 263]. Correspondingly, any perturbations that alter the levels or activity of Angiotensin Converting Enzyme (ACE), responsible for the conversion of angiotensin I to angiotensin II, also play a role in hypertensive remodeling and atherosclerosis [264].

Angiotensins include four main molecules (-I, -II, -III, and -IV) that bind and activate two isoforms of G-protein coupled receptors, the AT₁ and AT₂ [265]. AT₁ receptors can be further sub-classified as AT_1A or AT_1B, each with a unique tissue distribution [266, 267]. The AT₁ receptor appears to induce vascular remodeling when activated by angiotensin II [262, 268]. The AT₂ receptor is typically less abundant than the AT₁ except in developing tissues [268]. Stimulation of AT₂ receptors can inhibit proliferation and induce cellular differentiation [269]. The AT₂ receptor also can stimulate NO production and cGMP increases in the kidneys, especially during sodium depletion [270]. Tissue distributions of AT₁ and AT₂ receptors are highly heterogeneous, but both receptors can be expressed on vascular endothelia where they generally exert opposing effects [271]. Similarly, the AT₁ and AT₂ receptors also have opposing actions on angiotensin II mediated regulation of circulating blood volume and pressure [268, 272]. These effects can involve interactions among angiotensin II receptors, and the mineralocorticoid receptors that bind and respond to aldosterone [273]. Local inflammation can enhance the ability of angiotensin II to induce vascular remodeling [274]. Typically, angiotensin II stimulates expression of PDGF and TGF-β through activation of AT₁ receptors [275], and can increase eNOS and NO release in fetoplacental artery endothelial cells [276]. Angiotensin II can also transactivate certain tyrosine kinase receptors, including those that mediate responses to PDGF [262, 277].

One of the most important effects of AT₁ activation is increased formation of reactive oxygen species (ROS) [278]. These ROS molecules, which may originate from membrane-bound NADPH oxidase or mitochondrial synthesis [279], can induce vascular smooth muscle hypertrophy, hyperplasia, and migration [280, 281]. Activation of AT₁ by angiotensin II can increase the expression and activity of membrane-bound NADPH oxidase, and thereby stimulate ROS production [275, 278]. Increases in ROS can, in turn, have many effects including reaction with NO leading to decreased NO bioavailability. In turn, loss of NO can enhance the effects of angiotensin II on smooth muscle growth by upregulating AT₁ receptors, and can increase expression of endothelial ACE and ET-1 [282]. Angiotensin II can also increase HIF-1α gene expression and protein stability via a ROS-dependent mechanism [283, 284].

Numerous physiological and pathological perturbations influence the levels and cardiovascular effects of the angiotensins. Angiotensin II can be induced by VEGF, resulting in a positive feedback loop, in which the increased angiotensin II activates AT₁ receptors that further increase expression of HIF-1, VEGF, and VEGF receptors leading to additional increases in angiotensin II [285]. Hypoxia can alter AT₁ expression through mechanisms that appear highly sensitive to history and context; hypoxia has been reported both to increase [286] and decrease [287] AT₁ expression in vascular smooth muscle. During hypertension, the effects of angiotension II can be modulated by the simultaneous actions of FGF-2, resulting in enhanced stimulation of smooth muscle hypertrophy, proliferation and remodeling in cerebral but not extracerebral arteries. Conversely, angiotensin II can stimulate FGF-2 synthesis, and thereby amplify its effects on hypertension-induced cerebrovascular remodeling [288]. How angiotensin II contributes to hypoxic cerebrovascular remodeling remains unstudied, particularly in the immature cerebral circulation.

Catecholamines

Catecholamines serve important roles as neurotransmitters in both the central and peripheral nervous systems [289]. Aside from their well-documented effects on post-synaptic G-protein coupled receptors, both norepinephrine (NE) and serotonin (5-HT) can exert trophic effects on smooth muscle. These effects were recognized for NE in the late 1970s when it was observed that sympathetic denervation caused a relative atrophy and thinning of rabbit cerebral arteries [290, 291]. Subsequent studies furthered these findings and documented the ability of adrenergic perivascular nerves to stimulate phenotypic transformation in vascular smooth muscle [292] through activation of α_1A adrenergic receptors by NE [293]. Because chronic hypoxia can depress NE content and stimulation-evoked release [230, 294], chronic hypoxia should also attenuate the trophic influence of NE on cerebrovascular smooth muscle growth and differentiation. In addition, chronic hypoxia appears to depress NO release by perivascular nitrergic nerves [230], which should further compromise adrenergic vasotrophic stimulation of cerebrovascular growth and differentiation. Given that the adrenergic neuroeffector apparatus is functionally immature in fetal cerebral arteries [295], these results raise the possibility that cerebrovascular maturation relies on increasing trophic support from the adrenergic perivascular innervation. In turn, if chronic hypoxia inhibits the functional maturation of the adrenergic perivascular innervation, then the functional effects should be similar to adrenergic denervation in the fetal cerebral circulation. This hypothesis awaits experimental evaluation.

The other main neurotransmitter catecholamine with trophic effects is serotonin. This molecule can act through a broad variety of G-protein-coupled receptors [296, 297] that are heterogeneously expressed by both the smooth muscle and endothelium of virtually all blood vessel types [298]. In the pulmonary circulation, 5-HT can increase vascular permeability and induce smooth muscle proliferation. These effects appear to be mediated through activation of 5-HT_1B receptors and subsequent stimulation of ROS production [299]. Pathological increases in the expression of serotonin transporters (5-HTT or SERT) appear to enhance the proliferative, ROS-dependent effects of 5-HT on pulmonary smooth muscle [296, 300]. Some mitogenic effects of 5-HT, however, may be attributable to increased prostaglandin synthesis [297]. For example, 5-HT can stimulate prostacyclin production in aortic smooth muscle [301]. Prostacyclin, in turn, can stabilize HIF-1 through attenuation of ROS production [62] and both prostacyclin and PGE2 can increase expression of Ang2 [157]. Stimulation of prostacyclin receptors can upregulate smooth muscle cell contractile markers reflecting a shift from synthetic to contractile phenotype [30]. These effects of 5-HT may be more pronounced in older individuals [302]. Owing to the ability of estradiol to potentiate the proliferative effects of 5-HT, these effects can be more pronounced in females than in males and may contribute to the higher incidence of pulmonary arterial hypertension observed in women [303].

In relation to hypoxic vascular remodeling, hypoxia can increase mRNA for 5-HT and thereby enhance smooth muscle proliferation [304]. Adenosine, whose concentrations are elevated by hypoxia, can potentiate the proliferative effects of 5-HT by enhancing expression of the 5-HT transporter. This effect leads to internalization of 5-HT and increased ROS production, which contributes to the mitogenic effects of 5-HT on smooth muscle [304]. In contrast to other vasotrophic factors, hypoxia appears to have little effect on the expression of 5-HT receptors and their artery-size dependent patterns of expression [305]. It remains possible, however, that the perivascular serotonergic cerebrovascular innervation could be modulated by chronic hypoxia, as suggested for the adrenergic innervation. Because the serotonergic innervation is completely intracranial [306], it is not surgically possible to perform a denervation and observe the resulting effects on cerebovascular growth, differentiation, and function. Confirmation of a vasotrophic role for the serotonergic cerebrovascular innervation must await the development of alternative experimental approaches.

Purines

As a class, the purines couple tissue metabolic activity to vascular growth, proliferation, and contraction through actions on three main classes of G-protein coupled purinergic receptors (P1, P2X, and P2Y) [307]. Adenosine can activate four P1 receptors (A₁, A_2A, A_2B, and A₃) and also the P2X₁ receptor. ADP can activate both P2X and P2Y receptors [308]. ATP can bind and activate P2X₁ and P2Y receptors [308–311]. Together, the purines help regulate endothelial and smooth muscle proliferation, migration, and apoptosis and thereby contribute significantly to many patterns of vascular remodeling [310]. ATP, released by perivascular nerves and endothelial cells, can promote mitogenesis in vascular smooth muscle [312]. In relation to regulation of smooth muscle phenotype, synthetic smooth muscle tends to express P2Y₁ and P2Y₂ receptors more than P2X₁, whereas in contractile smooth muscle P2X₁ abundance predominates over that of the P2Y isoforms [29, 312]. This pattern raises the important question: are patterns of P2X and P2Y receptor expression a cause, or a consequence, of phenotypic transformation in smooth muscle? ADP can also induce proliferation and migration of endothelial cells, and can activate A₂ receptors to inhibit proliferation of smooth muscle cells [312]. In addition, ADP acts synergistically with PDGF, TGF-β, among others to induce VSM proliferation [310, 312]. Extracellular adenosine can contribute to pulmonary vascular remodeling via A_2A receptors, and extracellular actions of both ATP and adenosine can stimulate endothelial apoptosis and act through A₂ receptor, a P1 receptor subtype, to inhibit SMC proliferation [312, 313]. Hypoxia can inhibit ATP production due to decreased oxygen availability. On the other hand, hypoxia increases adenosine levels and thereby amplifies the proliferative effects of adenosine. For example, hypoxic increases in adenosine activate endothelial A_2A and A_2B receptors and stimulate EC proliferation [311, 312]. Importantly, A_2B receptor stimulation can also increase VEGF mRNA to promote angiogenesis [312]. Through activation of P2 and A_2A receptors, adenosine can also promote NO release and thereby activate NO-dependent influences on smooth muscle growth and differentiation [310, 311]. Hypoxia further potentiates these effects of adenosine by inhibiting the abundance and activity of adenosine kinase, the enzyme responsible for recycling of adenosine through conversion into AMP [314]. This effect is mediated by HIF-1α binding to HREs, which depresses transcription of the adenosine kinase gene [314]. As a group, the purines are important mediators of the coupling between oxygen dependent metabolic activity and vascular function. The importance of these mechanisms in the cerebral circulation remains largely unstudied in all age groups. Owing to the common therapeutic use of agents such as dipyridamole that alter circulating purine levels and actions [315], the potential vasotrophic effects of such treatments urge caution.

FUTURE DIRECTIONS

The past decade has ushered in a revolution in the understanding of vascular biology. The classical view of blood vessels as static, homogeneous structures has slowly yielded to the more contemporary view of the vascular wall as a highly dynamic and heterogeneous tissue with multiple cell types undergoing regular phenotypic transformation. The extent and character of these transformations are governed by a growing list of vasotrophic factors that continuously modulate vessel structure and function to support tissue growth and metabolic demand. The vasotrophic factors involved include not only the classical receptor tyrosine kinase ligands such as VEGF, PDGF, angiopoietins and FGF, but also a diverse category of smaller multifunctional molecules that influence smooth muscle growth and proliferation independent of receptor tyrosine kinases. This category includes TGF-β, nitric oxide, endothelin, angiotensin II, catecholamines, and purines. These non-classical vasotrophic factors appear to help fine-tune vascular composition and reactivity to meet the demands of tissue growth, development, and physiological stress. As seen repeatedly, the expression of these vasotrophic factors can be heterogeneous among various tissue types and vascular beds to ensure a close coupling between metabolic supply and demand. These fundamental differences in oxygen requirements for metabolic homeostasis among various tissues imply different susceptibilities to hypoxic insults. Consequently, both functional and structural adaptations of the vasculature are also organ specific. These mechanisms integrate to assure that blood flow and metabolic demand are closely matched in all vascular beds, especially under environmental stresses such as hypoxia. From this perspective, one of the most promising future endeavors will be to better understand the basic principle of “excitation-transcription coupling”, as introduced by Wamhoff [316]. This idea advances the notion that the same calcium transients that initiate muscle contraction simultaneously help activate key transcription factors, such as myocardin [317, 318], that drive expression of genes coding for critical proteins required for contraction. In this manner, contractile stimuli produce both short-term and long-term effects that serve to “condition” the blood vessels involved. How these signals integrate with other vasotrophic signals, growth factors, and pathogenic stimuli remains an exciting arena for future investigation.