Nearly all published articles on hypertension and race state that African Americans have the highest prevalence in the world. The 2006–2008 National Health and Nutrition Examination Survey data show that African American men and women aged 20 yr and older have a hypertension prevalence of 40% and 42%, whereas Caucasian men and women have a hypertension prevalence of 30% and 28% (18). The mechanisms underlying racial disparities in cardiovascular disease (CVD) are multifactorial. Differences in endothelial cell (EC) responses to stimuli could play an important role in the development of endothelial dysfunction, hypertension, and, therefore, CVD. Although only a handful of studies have been conducted, they consistently show that African Americans have reduced endothelial function compared with that in Caucasians (29).
A major problem with research on the racial disparity in endothelial dysfunction is that it is conducted at the organ or physiological system level but not at the cellular level. Because of this large gap between in vivo physiology and in vitro mechanistic research relative to racial disparities in endothelial dysfunction, there continues to be only speculation about mechanisms of endothelial dysfunction in African Americans. This lack of knowledge about the EC biology of African Americans severely impedes advances in finding optimal preventive and treatment strategies that target African Americans. Our research focuses on understanding racial disparities in endothelial function at the cellular level, if they exist.
The vascular endothelium is the monolayer of EC lining the blood vessels. Current thought about endothelial dysfunction includes a pro-oxidant, proinflammatory, prothrombotic, and apoptotic EC state that creates an arterial wall environment favoring the initiation and maintenance of high blood pressure and atherosclerosis. A quote by Le Brocq et al. (21) from a comprehensive article on endothelial dysfunction highlights the fact that there can be different types of endothelial dysfunction, and the best therapeutic plan may vary by type: “It is essential for researchers first to identify the type of endothelial dysfunction that applies to the disease of interest before deciding what therapeutic approach might be beneficial.”
Intravascular laminar shear stress (LSS) is a major physiological stimulus that can affect the phenotype of the endothelium profoundly (25). Our laboratory uses various levels of in vitro LSS to simulate different physiological blood flow conditions. For example, we use high physiological arterial levels of LSS as an exercise mimetic EC model to better understand the endothelial cellular and molecular events related to endothelial adaptations to exercise.
Figure 1 shows our conceptual model of proposed differences in EC status and responses to LSS in African Americans and Caucasians. Our working hypothesis is that EC obtained from African Americans exhibit greater levels of oxidative stress and inflammation compared with EC obtained from Caucasians. Furthermore, we hypothesize that African American EC demonstrate greater reductions in oxidative stress and inflammation after exposure to an in vitro exercise mimetic, LSS, compared with Caucasian EC. The data we present herein are, for the most part, the first of their kind. Thus, much more research is required to test these hypotheses fully.
ENDOTHELIAL DYSFUNCTION AND AFRICAN AMERICANS
Endothelial activation is an early step in endothelial dysfunction characterized by a disturbance of the regulation of vessel tone and the maintenance of a vascular environment that favors coagulation, oxidative stress, and inflammation. Endothelial dysfunction precedes and predicts the development of atherosclerotic disease in normal coronary arteries after cardiac transplant (11) and is a prognostic indicator of cardiovascular events after statistical adjustment for other CVD risk factors (43).
Approximately 10 studies have been conducted that assessed endothelial function in different races, and they consistently show reduced function in African Americans (29). This apparent predisposition for endothelial dysfunction likely contributes to their high prevalence of hypertension and hypertension comorbidities.
ENDOTHELIAL SHEAR STRESS
The endothelium senses both chemical and mechanical stimuli, integrates the signals, and transduces the signals across the membrane and into the EC (2). Intravascular shear stress is the tangential friction force exerted by the flowing blood on the vessel wall. The endothelium responds to a sudden increase in shear stress within milliseconds. This immediate response is followed within a few hours by changes in the regulation of a subset of genes that are expressed in EC. Thus, a critical feature of vascular endothelium is its phenotypic plasticity induced by hemodynamic cues. Mechanotransduction describes the interaction between biomechanical forces and EC function. Therefore, the mechanical forces acting on the luminal side of EC cause deformation of the EC that is transmitted through the cytoskeleton to the nucleus.
The type and magnitude of shear stress has an important role in long-term preservation of the structure and function of the blood vessel. Malek et al. (25) delineated the normal magnitudes of shear stress in veins, arteries, and in low-shear and high-shear pathologic states. Normal arterial shear stress levels range from 10 to 70 dynes·cm−2. Lower levels (<4 dynes·cm−2) stimulate an atherogenic phenotype, whereas levels of 15 dynes·cm−2 and higher induce an atheroprotective phenotype. Studies have shown that a prolonged (24-h) high physiological level of LSS profoundly alters EC phenotype by modifying the gene profile, creating an environment that is antiinflammatory, antioxidant, and antiapoptotic (25).
It is thought that the elevated vascular shear stress caused by aerobic exercise underlies many of the fundamental beneficial cardiovascular adaptations to aerobic exercise training. A substantial portion of the CVD risk reduction associated with exercise training cannot be explained entirely by changes in conventional CVD risk factors (32). It has been suggested that direct effects of exercise on the vessel wall may account for some of the remaining risk factor reduction gap (13).
A large body of work has shown that moderate-intensity aerobic exercise training improves endothelial function in animals and humans with and without CVD risk factors. It seems that endothelial function changes occur early (2–4 wk) in the exercise training program and may be a transient phenomenon because some long-term (>16 wk) aerobic training studies show a return to initial levels of endothelial-dependent dilation with vascular wall structural remodeling of vessel caliber (33,34). Furthermore, resistance and conduit vessels have a different time course of adaptation. Nonetheless, in individuals with diminished endothelial-dependent dilation, there is consistency with respect to the beneficial adaptations of the endothelium with aerobic exercise training. One question we had was because African Americans tend to have a reduced endothelial function compared with that in Caucasians, then would the two groups’ EC respond differently to an in vitro application of LSS that causes adaptations of EC that are consistent with in vivo endothelial function adaptations with exercise training?
DO ENDOTHELIAL NITRIC OXIDE SYNTHASE AND NITRIC OXIDE RESPOND DIFFERENTLY TO LSS IN AFRICAN AMERICAN AND CAUCASIAN ENDOTHELIAL CELLS?
The relative function of endothelial nitric oxide (NO) synthase (eNOS), the bioactivity of NO, and the consequent NO-dependent dilation are considered synonymous with the degree of endothelial function. A possible reason for reduced endothelial function in African Americans compared with that in Caucasians could be reduced eNOS protein and NO production. In addition, given the tendency for reduced endothelial function in African Americans, it would be important to understand the capacity for adaptability of their EC to exercise training. Therefore, we recently conducted a parallel EC culture experiment using African American and Caucasian human umbilical vein EC (HUVEC) to address this issue (9).
The HUVEC were exposed to 24 h of 5 dynes·cm−2 of LSS to simulate an atherogenic type of LSS, 24 h of 20 dynes·cm−2 of LSS to simulate a moderate exercise intensity level of LSS, and 0 dyne·cm−2 (static control conditions) using a custom cone and plate viscometer (9). For details about the methodology, please refer to the article by Feairheller et al. (9). The Table summarizes directional changes in all endothelial factors in response to LSS in African American and Caucasian HUVEC.
Under static basal conditions, African American HUVEC exhibited significantly greater eNOS protein expression levels compared with Caucasian HUVEC. Greater eNOS protein in the basal condition in African Americans would suggest the potential for greater NO production, although eNOS activation was not assessed in our study. This does not support the evidence for reduced endothelial function in African Americans. It is possible that NO production can be high but the NO bioavailability can be compromised, as is the case when oxidative stress levels also are elevated within the EC.
Interestingly, we also found that the African American HUVEC had significantly greater expression levels of inducible NOS (iNOS) compared with those in Caucasian HUVEC. Our results were similar to those of Kalinowski et al. (17) whose study also used African American and Caucasian HUVEC and found greater eNOS protein expression and NO levels in African American HUVEC. However, our study extended the results of Kalinowski et al. (17) by showing that basal expression of iNOS also was greater in African American HUVEC.
Greater iNOS expression may be very important in revealing the African American EC phenotype. This iNOS isoform is involved in immune response and produces large amounts of NO as a defense mechanism. The iNOS is up-regulated by different stimuli, especially inflammatory cytokines (16). Thus, greater iNOS expression may indicate an unhealthy or activated EC.
In response to LSS, we showed that eNOS protein expression was increased significantly in both African American and Caucasian HUVEC at 5 and 20 dynes·cm−2. Also, relative to 5 dynes·cm−2, eNOS protein expression significantly increased in both African American and Caucasian HUVEC at 20 dynes·cm−2 (Fig. 2). Relative to static controls, HUVEC from both African American and Caucasians increased total NO production when exposed to LSS at both 5 and 20 dynes·cm−2 (Fig. 2). Where there was a significant difference between HUVEC race was in NO production between 5 and 20 dynes·cm−2 of LSS; NO production significantly increased only in African American HUVEC. This finding has relevant physiological implications because 5 dynes·cm−2 of LSS can be considered a resting arterial level, whereas 20 dynes·cm−2 of LSS can be considered a level achieved during moderate-intensity aerobic exercise. The implications of these findings are that the adaptability of EC relative to eNOS and NO seems to be similar in the two racial groups.
Different magnitudes of LSS (i.e., 5 and 20 dynes·cm−2) could be thought of as different exercise intensities. A human study previously showed the exercise intensity–dependent nature of endothelial adaptations to exercise training. Goto et al. (12) assessed forearm endothelium-dependent dilation in healthy men after 12 wk of cycle ergometry training at 25% (mild), 50% (moderate), and 75% (high) of maximal oxygen consumption. The study found that only moderate-intensity exercise training increased endothelial-dependent dilation significantly. Neither low- nor high-intensity training significantly increased endothelial-dependent dilation. Our findings also show the LSS magnitude–dependent nature of changes in eNOS and NO.
OXIDATIVE STRESS AND ENDOTHELIAL CELLS
Oxidative stress plays a critical role in the pathology and progression of CVD and hypertension. Reactive superoxide (O2 -) primarily is produced by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Within the vascular wall, increased oxidative stress, as evidenced by higher O2 - levels, alters blood flow regulation, membrane lipid oxidation, protein oxidation, platelet aggregation, leukocyte adhesion, and controls cellular growth. Many extensive review articles have highlighted the complex association between reactive oxygen species (ROS) and their contributions to altered physiology and subsequent pathologies (35).
Within the body, the three primary antioxidant defense enzymes are superoxide dismutase (SOD), catalase, and glutathione peroxidase. The SOD enzyme serves as the main protective antioxidant scavenging enzyme, where under normal physiological conditions, the dismutation of O2 - by SOD yields hydrogen peroxide (H2O2). Three main isoforms of the SOD enzyme exist: manganese SOD (MnSOD/SOD2) located in the mitochondria, copper/zinc SOD (CuZnSOD/SOD1) located in the cytoplasm, and extracellular SOD (EcSOD/SOD3) located in the extracellular space. In the case of heightened NOX activity and O2 -overproduction, O2 - reacts with NO to produce peroxynitrite (ONOO-), thus reducing NO bioavailability. When concentrations of O2 - and NO are high, formation of ONOO- is favored because the rate of this diffusion-limited reaction is three times faster than the dismutation of O2 - by SOD (19).
The NOX enzyme complex is composed of a membrane-bound cytochrome, which includes subunits gp91phox and p22phox, and a cytosolic component composed of subunits p47phox, p67phox, and the G protein Rac. NOX-mediated free radical production has been associated with the pathophysiology of endothelial dysfunction, inflammation, CVD, and angiogenesis. It has been reported that in primary endothelial cultures, NOX2 and NOX4 serve as primary sources of NOX-mediated oxidative stress (36).
It has been shown that exercise training modifies the expression of NOX. In an in vivo study by Adams et al. (1), left internal mammary artery rings sampled during bypass surgery from 45 patients randomized to either an exercise training or an inactive control group were assessed for messenger RNA (mRNA) expression of NOX subunits, NOX activity, and ROS production. Exercise training significantly lowered the expression of gp91phox, p22phox, and NOX4. NOX enzymatic activity and ROS generation were significantly lower in the exercise training group compared with those in the control group. Exercise training also resulted in improved endothelial function.
Very little in vitro research has been conducted examining possible differences between cell lines of different races. Wei et al. (42) conducted a microarray study to assess basal gene expression in an attempt to explain racial health disparities. They concluded that genetic influences stemming from ancestral continent of origin could impact on EC biology and thereby contribute to disparity of vascular-related disease burden among African Americans. Kalinowski et al. (17) found that African American HUVEC exhibited up-regulated eNOS and NOX subunit expression of p67phox, p47phox, and p22phox relative to those in Caucasian HUVEC.
Duerrschmidt et al. (8) used cultured HUVEC of unknown racial origin to quantify the expression of specific NOX subunits in response to LSS. They reported reduced mRNA expression of gp91phox and p47phox after 24 h of LSS at 30 dynes·cm−2. Because in vitro LSS is hypothesized to simulate the shear stress conditions elicited by aerobic exercise in vivo, this reported in vitro downregulation of NOX activity may be an important contributor to the lower oxidative stress levels often observed after exercise training in human subjects. However, no study has ever investigated the possibility that there may be an influence of race on NOX subunit response to LSS.
For us to pursue potential race differences in EC oxidative stress, we first sought to examine whether basal oxidative-stress markers that typically are measured in humans also differ by race in cell culture (10). For detailed methodology, please refer to the article by Feairheller et al. (10). For the EC experiments, African American and Caucasian HUVEC were cultured in parallel. The supernatant from confluent HUVEC was removed and immediately stored at −80°C until assay. Cell lysate was collected by fractionation, and cell culture media samples were collected.
We found heightened oxidative stress in the African Americans both in vitro and in vivo. We found no difference in plasma NO levels between the African American and Caucasian young adults. African American adults had significantly greater plasma SOD activity compared with that in Caucasian adults. Total antioxidant capacity (TAC) provides a relative cumulative measure of all the antioxidants present in the plasma and body fluid and inherently is a measure of the ability to scavenge H2O2. African American adults also had significantly greater plasma TAC levels compared with those in Caucasian adults. Confirming this heightened oxidative stress, we found significantly greater plasma protein carbonyl levels in African American adults compared with those in Caucasian adults. Relative to the HUVEC, we found significantly lower SOD activity in African American compared with those in Caucasian HUVEC. To further clarify the antioxidant potential of the HUVEC, we found that African American HUVEC tended to have lower SOD1 activity. Further analysis of protein expression of SOD2 showed that African American HUVEC exhibited significantly lower basal SOD2 protein expression.
In most tissues, SOD3 exists in very small amounts, but in the circulation, it can represent up to one half of the total SOD activity. Therefore, in controlled cell culture conditions, EC lysates would not have the same inherent percentages of SOD3. In HUVEC, we found that African Americans had significantly lower levels of total SOD activity, whereas in human plasma, we found that African American adults had significantly higher SOD activity when compared with those in Caucasians. This difference could be attributed to the large contributions from SOD3 to the total SOD measured in plasma from the African American adults.
In summary, we found evidence of a heightened oxidative stress environment both in vivo and in vitro in African Americans. Given the often observed relationship of oxidative stress with various chronic diseases, the potential implication is that interventions aimed at reducing oxidative stress and/or increasing antioxidant capacity within the vascular wall may be especially important in African Americans. Furthermore, these observational results suggest that the race of the cell model may be important to consider in the context of in vivo clinical research.
DO OXIDATIVE STRESS MECHANISMS IN AFRICAN AMERICAN AND CAUCASIAN ENDOTHELIAL CELLS RESPOND DIFFERENTLY TO LSS?
In our parallel EC culture experiments using African American and Caucasian EC (9,10), we found greater expression levels of oxidative stress proteins during basal conditions (static) in African American HUVEC. African American HUVEC exhibited significantly greater p47phox, p22phox, NOX2, and NOX4 protein expression levels compared with Caucasian HUVEC (Fig. 3).
We hypothesized that a high level of LSS would be beneficial because it would decrease NOX subunit protein expression. In our study, relative to static controls, p47phox protein expression levels significantly decreased with 20 dynes·cm−2 LSS only in African American HUVEC (9). Relative to static controls, NOX4 protein expression significantly decreased only in the African American HUVEC with 5 dynes·cm−2 of LSS (Fig. 4), but with 20 dynes·cm−2 of LSS, NOX4 expression significantly decreased in both African American and Caucasian HUVEC (Fig. 4). LSS had no effect on NOX2 protein expression in HUVEC from either race. In response to LSS, SOD2 protein expression significantly increased in African American HUVEC under the 5- and 20- dynes·cm−2 conditions. Again, only the African American HUVEC had a significant increase in total SOD activity under the 5- and 20-dynes·cm−2 conditions compared with static conditions. LSS had no effect on either SOD2 protein expression or total SOD activity in the Caucasian HUVEC. Compared with static conditions, the African American HUVEC had greater TAC responses to 5 and 20 dynes·cm−2 of LSS. The Caucasian HUVEC did not respond to 5 dynes·cm−2 but did achieve significantly greater TAC levels at 20 dynes·cm−2 compared with those in static conditions.
Taken together, p47phox and NOX4 were more responsive to LSS in African American EC, and SOD2 protein expression significantly increased only in African American EC under the 5- and 20-dynes·cm−2 conditions. Studies have found that p47phox is required for NADPH oxidase activity and ROS production in EC (23). It has been shown that in mice lacking p47phox, the blood pressure response to angiotensin II was blunted compared with wild-type mice, suggesting a role for p47phox and NOX activation in the blood pressure increase caused by angiotensin II (20). It has been reported that, in primary EC cultures, NOX4 serves as a primary source of oxidative stress (36). This NOX4 isoform also has been suggested to directly produce large amounts of the oxidant H2O2 (22). This information has important implications. Given that African Americans may have an EC phenotype characterized by heightened oxidative stress, it is important to show that a nonpharmacologic intervention could be effective in reducing this vascular oxidative stress burden that could improve endothelial function and reduce the risk of hypertension in this high-risk group.
INFLAMMATION AND ENDOTHELIAL CELLS
Inflammation directly damages the endothelium by activating the EC and indirectly by inducing oxidative stress. Epidemiologic data support the existence of an association between different inflammatory markers and high blood pressure. One such inflammatory marker is C-reactive protein (CRP). CRP is considered to be an important marker of both subclinical chronic vascular inflammation and increased cardiovascular risk (30). CRP is an acute-phase protein that is produced by hepatocytes and is regulated by cytokines, mostly interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) (3). CRP also stimulates monocytes to increase the release of other cytokines and the expression of the adhesion molecules by EC (27). In general, studies have shown a graded risk across the measured ranges of high-sensitivity CRP (hs-CRP). Increased levels of proinflammatory markers, such as hs-CRP, have been found in hypertensive individuals, even after statistical adjustment for potential confounding factors. Elevated hs-CRP levels also have been shown to be predictive of the development of hypertension in prehypertensive and normotensive patients (7). We consider a weakness of the studies previously mentioned is that they are limited to Caucasian North American or European populations except for one study that included Japanese-American men in the Honolulu Heart Study (31).
African Americans tend to have higher levels of inflammatory markers compared with those in Caucasians, which may be related to their greater risk of CVD-related morbidity and mortality. We recently have shown in a cohort of African American prehypertensives (systolic blood pressure, 120–139 mm Hg and diastolic blood pressure, 80–89 mm Hg) that there is low-grade systemic inflammation, as indicated by hs-CRP levels (38). In fact, their hs-CRP levels put them in the highest CVD risk category (28).
Nonpharmacological and pharmacological approaches tend to reduce vascular inflammation in patients with diabetes and hypertension. A number of studies have assessed the effects of exercise training on CRP in coronary artery disease patients (40). In general, these studies indicate that exercise training reduces inflammation, independent of changes in obesity. Because the endothelium is a source of inflammatory cytokines, it is likely that an improvement in EC health could be a primary reason for the improvement in chronic inflammation with exercise training.
In response to proinflammatory cytokine stimulation, EC undergo functional changes that include an increase in the expression of adhesion molecules and induction of procoagulant activity. Proinflammatory cytokines, such as TNF-α, activate nuclear factor-κB transcription factors that regulate vascular inflammation. In addition, CRP is not simply a marker of chronic inflammation but also has direct effects on EC by down-regulating eNOS transcription, destabilizing eNOS mRNA, and decreasing basal and stimulated NO release (39). TNF-α decreases eNOS mRNA expression level by shortening its half-life (44). Secondarily, increased EC production of O2 - and other free radicals during inflammation can reduce NO bioavailability. Evidence shows that chronic high physiological levels of LSS, which are consistent with atheroprotection, suppress inflammation in EC (26).
ARE THERE RACIAL DIFFERENCES IN ENDOTHELIAL MICROPARTICLES AS MARKERS OF ENDOTHELIAL CELL DAMAGE?
The physiological measurement of endothelial function does not provide specific information about the biological status of EC. Cytokine-mediated endothelial dysfunction seems to be a predominant mechanism underlying hypertension in African Americans. Endothelial microparticles (EMP) are submicroscopic membranous particles that reflect the functional state of the parental EC (14). EMP are released from activated or apoptotic parent EC when stimulated by proinflammatory cytokines, oxidative stress, or infectious agents (37). They carry a subset of membrane proteins and phospholipids, or markers, of their parent EC, including those induced by activation, apoptosis, or oxidative stress. EMP expressing constitutive surface markers such as CD31 (platelet EC adhesion molecule) are increased in injury and/or apoptosis (24). EMP levels are increased in pathological conditions such as diabetes, hypertension, metabolic syndrome, hypercholesterolemia, and thrombosis. Jenkins et al. (15) recently published an article reporting that previous exercise prevents postprandial-induced oxidative stress in circulating angiogenic cells. They found that the attenuated increases in oxidative stress by previous exercise was associated with increased antioxidant gene expression and reduced EMP compared with those in the control trial. Thus, the level of EMP is emerging as a novel direct marker of EC impairment mediated by activation and apoptosis.
We recently published a study that determined EMP responses to the inflammatory cytokine, TNF-α, and the antioxidant, SOD, in HUVEC obtained from African Americans and Caucasians (5). TNF-α is a primary inflammatory cytokine that is synthesized in several cell types involved in vascular inflammation including EC. Intra-arterial TNF-α has been shown to cause acute local vascular inflammation that is associated with impaired endothelium-dependent relaxation (6).
In our study, EMP were measured under four conditions: control (basal), TNF-α, SOD, and TNF-α + SOD. Culture supernatant was collected for EMP analysis by flow cytometry and IL-6 assay by enzyme-linked immunosorbent assay. IL-6 protein expression was assessed by Western blot. We measured the cytokine IL-6 because it is produced by EC in response to TNF-α and stimulates the synthesis of several acute-phase reaction proteins, including CRP. IL-6 prolongs endothelial dysfunction, which may in turn lead to hypertension.
We showed that the EMP level significantly increased by 89% from basal levels in the African American HUVEC under the TNF-α condition compared with a nonsignificant increase of just 8% in the Caucasian HUVEC (Fig. 5). Compared with the EMP level under the TNF-α condition, the EMP level in the African American HUVEC was significantly lower under the SOD-only condition and under the TNF-α + SOD condition. Basal IL-6 concentrations were nearly twofold greater in the African Americans compared with those in the Caucasian HUVEC, whereas basal IL-6 protein expression was significantly greater in the African American cells (Fig. 6). These observational results suggest that African American HUVEC may be more susceptible to the injurious effects of the proinflammatory cytokine TNF-α. In addition, the results suggest that TNF-α–induced oxidative stress may play a greater role in EMP generation in African Americans compared with that in Caucasian HUVEC. Because we found that the EMP response to TNF-α differed by HUVEC race, we next wondered if high physiological levels of LSS would differentially affect EMP levels in African American and Caucasian EC.
DO AFRICAN AMERICAN AND CAUCASIAN ENDOTHELIAL CELLS SHOW DIFFERENTIAL EMP GENERATION RESPONSES TO LSS?
We conducted a similar LSS experiment and measured the generation of EMP in African American and Caucasian HUVEC. We hypothesized that because EMP are markers of EC damage, then low (5 dynes·cm−2) physiological levels of LSS (those that are atherogenic prone) would increase EMP levels and that high (20 dynes·cm−2) physiological levels of LSS (those that are atheroprotective) would decrease EMP levels. We found a tendency for a HUVEC race by LSS condition interaction with the Caucasian HUVEC, showing a substantial reduction in EMP generation, going from 5 to 20 dynes·cm−2 of LSS, whereas EMP generation was not altered in the African American HUVEC (Fig. 7). There was a significant main effect of LSS. There was no main effect of race. These results suggest that EMP are responsive to LSS, although only the Caucasian HUVEC showed significantly lower levels of EMP at 20 dynes·cm−2 compared with the 5-dynes·cm−2 condition. Indeed, much more research is required to elucidate the underlying mechanisms responsible for LSS-induced changes in EMP.
In summary, we have shown that African American HUVEC had a much greater EMP response to TNF-α and SOD, suggesting that there was greater EC damage and that it was likely that TNF-α–induced oxidation stress played a prominent role. High physiological levels of LSS are known to improve EC phenotype such that the EC is anti-inflammatory, antioxidative, and antiapoptotic. We have shown that there was a significant main effect of 20 dynes·cm−2 of LSS to reduce EMP levels; however, this effect primarily was caused by the Caucasian HUVEC. This resulted in a tendency for an interactive effect of HUVEC race and LSS.
Our working hypotheses are that EC obtained from African Americans exhibit greater levels of oxidative stress and inflammation compared with EC obtained from Caucasians and that African American EC demonstrate greater reductions in oxidative stress and inflammation after exposure to an in vitro exercise mimetic, LSS, compared with those in Caucasian EC. Nearly three decades of research by numerous independent laboratories have established clearly that the shear effects determined in vitro using aortic or venous EC of humans and animals well reflect the in vivo responses in both human and animals. Even after an EC demonstrates a particular phenotype during embryonic development, transdifferentiation may occur (41) because EC adapt to hemodynamic environmental cues (4). HUVEC yield reproducible biologic responses and demonstrate most of the same patterns of gene expression as EC from coronary artery and aorta.
The most important findings of our research relative to responses to LSS are related to oxidative stress. Relative to static controls, p47phox protein expression levels significantly decreased with 20 dynes·cm−2 LSS only in African Americans. Relative to static controls, NOX4 protein expression significantly decreased only in the African American HUVEC with 5 dynes·cm−2 LSS. Relative to NO production, only the African American HUVEC increased NO production at 20 dynes·cm−2 of LSS compared with 5 dynes·cm−2. We currently are conducting studies to elucidate the reasons for possible race-dependent responses to LSS. To this point, our data should be viewed as preliminary.
We recognize that human exercise training consists of intermittent bouts of exercise that occur over time and that our application of LSS occurs for 24 h. The reason we selected 24 h is because it is more than enough time for gene expression to occur and for protein levels to change. The adaptations in protein expression that occur after 24 h of LSS are consistent with the changes in endothelial function in humans and animals after exercise training. The findings we report here are from our initial sets of experiments aimed at addressing our hypotheses. Of course, subsequent experimental designs will use many different types of LSS perturbations, including intermittent application.
Taken together, our data suggest that African Americans seem to have an EC phenotype characterized by heightened oxidative stress, with the African American HUVEC exhibiting greater basal p47phox, NOX2, and NOX4 protein expression levels compared with those in Caucasian HUVEC. To compound this presumed heightened oxidative stress, the African American HUVEC had a reduced antioxidant capacity exhibiting lower SOD activity and SOD2 protein expression levels. We also showed that African American HUVEC exhibit potential for a heightened inflammatory phenotype. In addition, African American HUVEC exhibited greater basal iNOS protein expression levels, which could be caused by the greater oxidative stress and inflammation.
It may be that the fundamental mechanism underlying endothelial dysfunction in African Americans is EC inflammation, which can lead to increased oxidative stress. Interestingly, our African American subjects demonstrate an average hs-CRP concentration that puts them in the highest risk category (>3.0 mg·L−1) and also exhibit endothelial dysfunction, even when we analyzed data from only the prehypertensive African American group.
There is a very large group of literature describing observations of racial disparities in various diseases and conditions even when genetic admixture is present. Because of the observational nature of these studies, gaining insights into specific mechanisms that can be targeted for therapy cannot be determined. Even the studies that are conducted at the organ or physiological system levels are not able to identify the fundamental cellular mechanisms underlying the potential biologic causes of racial health disparities. Thus, progress in racial health disparities research is stymied.
Based on the few EC proteins that we have investigated to date, some seem to have race dependency in terms of their responses to LSS. Our future work will be directed at exploring specific inflammatory and oxidative stress pathways and their modifiers more deeply. Of course, EC genes that are shear stress responsive will be primary targets.
The authors thank Dr. Joon Young Park and Boa Kim of the Cardiovascular Genomics Lab at Temple University for their technical guidance and expertise.
This study supported by NIH/NHLBI Grant RO1 HL085497 (PI, M. Brown) and by NIH/NIA Grant KO1 AG019640 (PI, M. Brown).
1. Adams V, Linke A, Krankel N, et al.. Impact of regular physical activity on the NAD(P)H oxidase and angiotensin receptor system in patients with coronary artery disease. Circulation.
2005; 111: 555–62.
2. Barakat A, Lieu D. Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress. Cell Biochem. Biophys.
2003; 38: 323–43.
3. Baumann H, Gauldie J. The acute phase response. Immunol. Today.
1994; 15: 74–80.
4. Braddock M, Schwachtgen JL, Houston P, Dickson MC, Lee MJ, Campbell CJ. Fluid shear stress modulation of gene expression in endothelial cells. News Physiol. Sci.
1998; 13: 241–6.
5. Brown MD, Feairheller DL, Thakkar S, Veerabhadrappa P, Park JY. Racial differences in tumor necrosis factor-alpha–induced endothelial microparticles and interleukin-6 production. Vasc. Health Risk Manag.
2011; 7: 541–50.
6. Chia S, Qadan M, Newton R, Ludlam CA, Fox KA, Newby DE. Intra-arterial tumor necrosis factor-alpha impairs endothelium-dependent vasodilatation and stimulates local tissue plasminogen activator release in humans. Arterioscler. Thromb. Vasc. Biol.
2003; 23: 695–701.
7. Chrysohoou C, Pitsavos C, Panagiotakos DB, Skoumas J, Stefanadis C. Association between prehypertension status and inflammatory markers related to atherosclerotic disease: The ATTICA Study. Am. J. Hypertens.
2004; 17: 568–73.
8. Duerrschmidt N, Stielow C, Muller G, Pagano PJ, Morawietz H. NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells. J. Physiol.
2006; 576: 557–67.
9. Feairheller DL, Park JY, Rizzo V, Kim B, Brown MD. Racial differences in the responses to shear stress in human umbilical vein endothelial cells. Vasc. Health Risk Manag.
2011; 7: 425–31.
10. Feairheller DL, Park JY, Sturgeon KM, et al.. Racial differences in oxidative stress and inflammation: in vitro
and in vivo
. Clin. Transl. Sci.
2011; 4: 32–7.
11. Fish RD, Nabel EG, Selwyn AP, et al.. Responses of coronary arteries of cardiac transplant patients to acetylcholine. J. Clin. Invest.
1988; 81: 21–31.
12. Goto C, Higashi Y, Kimura M, et al.. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stress. Circulation.
2003; 108: 530–5.
13. Green DJ, O’Driscoll G, Joyner MJ, Cable NT. Exercise and cardiovascular risk reduction: time to update the rationale for exercise? J. Appl. Physiol.
2008; 105: 766–8.
14. Huang AL, Vita JA. Effects of systemic inflammation on endothelium-dependent vasodilation. Trends Cardiovasc. Med.
2006; 16: 15–20.
15. Jenkins NT, Landers RQ, Thakkar S, et al.. Prior endurance exercise prevents postprandial lipaemia-induced increases in reactive oxygen species in circulating CD31+ cells. J. Physiol.
2011; 589: 5539–53.
16. Kaku Y, Nanri H, Sakimura T, Ejima K, Kuroiwa A, Ikeda M. Differential induction of constitutive and inducible nitric oxide synthases by distinct inflammatory stimuli in bovine aortic endothelial cells. Biochim. Biophys. Acta.
1997; 1356: 43–52.
17. Kalinowski L, Dobrucki IT, Malinski T. Race-specific differences in endothelial function: predisposition of African Americans to vascular diseases. Circulation.
2004; 109: 2511–7.
18. Keenan NL, Rosendorf KAPrevalence of hypertension and controlled hypertension—United States, 2005–2008. Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report
2011; 60: 94–7.
19. Kojda G. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc. Res.
1999; 43: 562–71.
20. Landmesser U, Cai H, Dikalov S, et al.. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension.
2002; 40: 511–5.
21. Le Brocq M, Leslie SJ, Milliken P, Megson IL. Endothelial dysfunction: from molecular mechanisms to measurement, clinical implications, and therapeutic opportunities. Antioxid. Redox. Signal.
2008; 10: 1631–74.
22. Lee CF, Qiao M, Schroder K, Zhao Q, Asmis R. Nox4 is a novel inducible source of reactive oxygen species in monocytes and macrophages and mediates oxidized low density lipoprotein-induced macrophage death. Circ. Res.
2010; 106: 1489–97.
23. Li JM, Mullen AM, Yun S, et al.. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ. Res.
2002; 90: 143–50.
24. Mackay F, Loetscher H, Stueber D, Gehr G, Lesslauer W. Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J. Exp. Med.
1993; 177: 1277–86.
25. Male AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA.
1999; 282: 2035–42.
26. Partridge J, Carlsen H, Enesa K, et al.. Laminar shear stress acts as a switch to regulate divergent functions of NF-kappaB in endothelial cells. FASEB J.
2007; 21: 3553–61.
27. Pasceri V, Cheng JS, Willerson JT, Yeh ET. Modulation of C-reactive protein–mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. Circulation.
2001; 103: 2531–4.
28. Pearson TA, Mensah GA, Alexander RW, et al.. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation.
2003; 107: 499–511.
29. Perregaux D, Chaudhuri A, Rao S, et al.. Brachial vascular reactivity in blacks. Hypertension.
2000; 36: 866–71.
30. Ridker PM, Morrow DA. C-reactive protein, inflammation, and coronary risk. Cardiol. Clin.
2003; 21: 315–25.
31. Sakkinen P, Abbott RD, Curb JD, Rodriguez BL, Yano K, Tracy RP. C-reactive protein and myocardial infarction. J Clin Epidemiol
2002; 55: 445–51.
32. Thijssen DH, Maiorana AJ, O’Driscoll G, Cable NT, Hopman MT, Green DJ. Impact of inactivity and exercise on the vasculature in humans. Eur. J. Appl. Physiol.
2010; 108: 845–75.
33. Thijssen DH, Rongen GA, Smits P, Hopman MT. Physical (in)activity and endothelium-derived constricting factors: overlooked adaptations. J. Physiol.
2008; 586: 319–24.
34. Tinken TM, Thijssen DH, Hopkins N, Dawson EA, Cable NT, Green DJ. Shear stress mediates endothelial adaptations to exercise training in humans. Hypertension.
2010; 55: 312–8.
35. Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: what is the clinical significance? Hypertension
2004; 44: 248–52.
36. Van Buul JD, Fernandez-Borja M, Anthony EC, Hordijk PL. Expression and localization of NOX2 and NOX4 in primary human endothelial cells. Antioxid. Redox Signal.
2005; 7: 308–17.
37. Vanden Berghe W, Vermeulen L, De Wilde G, De Bosscher K, Boone E, Haegeman G. Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6. Biochem. Pharmacol.
2000; 60: 1185–95.
38. Veerabhadrappa P, Diaz KM, Kashem AM, et al.. Endothelial dysfunction in prehypertensive African Americans with masked hypertension. Am. J. Hypertens.
2011; 24: 1102–7.
39. Verma S, Wang CH, Li SH, et al.. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation.
2002; 106: 913–9.
40. Walther C, Mobius-Winkler S, Linke A, et al.. Regular exercise training compared with percutaneous intervention leads to a reduction of inflammatory markers and cardiovascular events in patients with coronary artery disease. Eur J Cardiovasc Prev Rehabil.
2008; 15: 107–12.
41. Wasserman SM, Mehraban F, Komuves LG, et al.. Gene expression profile of human endothelial cells exposed to sustained fluid shear stress. Physiol. Genomics.
2002; 12: 13–23.
42. Wei P, Milbauer LC, Enenstein J, Nguyen J, Pan W, Hebbel RP. Differential endothelial cell gene expression by African Americans versus Caucasian Americans: a possible contribution to health disparity in vascular disease and cancer. BMC Med.
2011; 9: 1–18.
43. Yeboah J, Crouse JR, Hsu FC, Burke GL, Herrington DM. Brachial flow–mediated dilation predicts incident cardiovascular events in older adults: the Cardiovascular Health Study. Circulation.
2007; 115: 2390–7.
44. Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ. Res.
1993; 73: 205–209.
Keywords:©2013 The American College of Sports Medicine
endothelial cell; shear stress; exercise; African American; oxidative stress; inflammation