Race-Specific Differences in Endothelial FunctionPredisposition of African Americans to Vascular DiseasesLeszek Kalinowski, MD, PhD; Iwona T. Dobrucki, MS; Tadeusz Malinski, PhDBackground—The prevalence of the endothelium-impaired function disorders, such as hypertension and diabetes mellitus,and the severity of their complications are considerably greater in blacks than whites. Evidence has accumulated thatsuperoxide (O2⫺) production and its interaction with nitric oxide (NO), yielding the strong oxidant peroxynitrite(ONOO⫺), play central roles in vascular pathophysiology. We hypothesized that the differences in endothelialNO/O2⫺/ONOO⫺metabolism may highlight the potential predisposition to endothelial dysfunction and cardiovascularcomplications prevalent in blacks.Methods and Results—Highly sensitive tandem electrochemical NO/O2⫺/ONOO⫺nanosensors were positioned in singlehuman umbilical vein endothelial cells (HUVECs) isolated from blacks and whites, and the kinetics of NO/O2⫺/ONOO⫺release were recorded in vitro. HUVECs were also analyzed by Western immunoblotting and enzyme activity assays forNAD(P)H-oxidase and endothelial NO synthase (eNOS). Compared with whites, HUVECs from blacks elicited reducedrelease of bioactive NO with an accompanying increase in the release of both O2⫺and ONOO⫺. The greater potencyof NO production because of eNOS upregulation in HUVECs from blacks is associated with a decrease in the NObioavailability. This is due to increased NO degradation by excess O2⫺produced primarily by 2 enzymatic sources:NAD(P)H-oxidase and uncoupled eNOS.Conclusions—Compared with whites, the steady-state NO/O2⫺/ONOO⫺balance in endothelial cells from blacks is keptcloser to the redox states characteristic for the endothelium-impaired function disorders. This may explain thedifferences in racial predisposition to the endothelium dysfunction during ongoing vascular disturbances with thehallmark of enhanced NO inactivation within the endothelium by oxidative stress. (Circulation. 2004;109:2511-2517.)Key Words: nitric oxide䡲endothelium䡲risk factors䡲African Americans䡲ethnic groupsIn the past few decades, the excess of hypertension anddiabetes mellitus among people of African descent (blacks)has been recognized as a substantial portion of the apparentblack health disadvantage. This is especially true for AfricanAmericans, who have one of the highest rates of hypertensionand diabetes mellitus in the world.1,2Cardiovascular compli-cations associated with these diseases, such as stroke andheart and renal failures, are responsible for the greater rates ofmortality in blacks compared with whites.Endothelium-derived NO is a physiological mediator ofnumerous cellular and organ functions. One important phys-iological role of NO is to protect the cardiovascular systemagainst pathophysiological insults. Besides being the mostpotent endogenous vasodilator, NO also inhibits smoothmuscle cell proliferation and migration, adhesion of leuko-cytes to the endothelium, and platelet aggregation. An im-pairment of the NO signaling pathway, ie, endothelial dys-function, is one of the earliest events in vascular diseases. Thereduced bioavailability of NO observed in the pathogenesis ofvascular diseases may occur by a reduction in NO synthesisand an increase in O2⫺generation. O2⫺reacts rapidly withNO, reducing NO bioactivity and producing ONOO⫺,astrong oxidant. The increased O2⫺production within endo-thelium accounts for a significant portion of the relative NOdeficit in the vascular diseases, including hypertension anddiabetes. In the majority of cases, the source of O2⫺excess isuncertain, although involvement of NAD(P)H-dependent oxi-dases, xanthine oxidase, cyclooxygenase, mitochondrial oxi-dases, and endothelial nitric oxide synthase (eNOS) andneuronal NOS have been suggested.3–6It should be noted thatthe net effect of the reaction between NO and O2⫺compro-mises reduction of concentration of both substrates as well asbiological effects of ONOO⫺itself. Peroxynitrite induces theoxidation of proteins, DNA, and lipids in vascular walls.Thus, increased production of a potentially deleterious me-tabolite, ONOO⫺, by eNOS may shift the balance betweenoxidative and reductive states of the endothelial cell and mayalter the beneficial effects of increasing NO activity. Modu-lation of either production or removal of NO and O2⫺isreflected in changes of ONOO⫺formation and makes fluctu-ations in their levels transient.Received October 13, 2003; de novo received December 12, 2003; accepted February 17, 2004.From the Department of Biochemistry, Ohio University, Athens. Dr Kalinowski was on sabbatical leave from the Department of Clinical Biochemistry,Medical University of Gdansk, and Laboratory of Cellualr and Molecular Nephrology of the Polish Academy of Science, Poland.Correspondence to Tadeusz Malinski, Department of Biochemistry, Ohio University, Athens, OH 45701. E-mail [email protected]© 2004 American Heart Association, Inc.Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000129087.81352.7A2511In this work, we electrochemically assayed7–9NO, O2⫺, andONOO⫺with modified electrodes into a tandem of NO/O2⫺/ONOO⫺nanosensors, for the first time allowing us tomeasure concurrently the molecules in real time in a singleendothelial cell. This approach is extremely favorable towardunderstanding the processes and mechanism of pathogenesisin vascular diseases at the molecular level. We sought todetermine whether the predisposition to vascular complica-tions in blacks implicates a reduction of NO bioavailability inendothelium. If so, what mechanism underlies the endotheli-um-determined race-specific diversity? We found that de-creased NO availability in blacks compared with whites isdue to excess O2⫺produced by NAD(P)H-oxidase that finallyyields enhanced formation of ONOO⫺after stimulation ofeNOS. This, in turn, leads to the eNOS uncoupling, whichproduces O2⫺and NO, and may very well contribute tooxidative stress and endothelial dysfunction.MethodsSubjects and Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolatedinto primary cultures from 12 white and 12 black female donors byClonetics and purchased as proliferating cells. All cell culture donorswere healthy, and none had pregnancy or perinatal complications.The clinical characteristics of the donors are reported in Table 1.None of the donors