Abstract
Sickle cell disease is fundamentally an inflammatory state, and endothelial activation and dysfunction have significant roles in the pathophysiology of this disease. In the last decade, research in the cardiovascular field has proven that the hormone aldosterone, canonically viewed as a regulator of renal electrolyte handling and blood pressure, also has direct, pro-inflammatory effects on the vascular endothelium that are independent of its classical effects. Excessive aldosterone is now known to cause microvascular damage, vascular inflammation, oxidative stress and endothelial dysfunction although the molecular mechanisms remain poorly understood (Brown, Hypertension 2008). In addition, aldosterone decreases endothelial cell production of nitric oxide and upregulates VCAM-1 and ICAM-1 production, leading to increased leukocyte-endothelial cell adhesion (Oberleithner, PNAS, 2007; Krug, Hypertension 2007). In animal models, aldosterone-mediated vascular injury in the brain, heart, and kidneys leads to stroke, myocardial injury, and renal damage (Marney, Clin Sci 2007). In addition, several large clinical trials have shown that aldosterone-antagonizing medications decrease mortality in patients with renal and heart failure, due in part to the blocking of the inflammatory vascular effects of this hormone (Pitt, N Engl J Med, 2003). Although the vascular effects of aldosterone are similar to those that occur in sickle cell disease, no published studies to date have investigated the possible interactions between aldosterone and sickle cell disease. Furthermore, the efficacy of aldosterone-antagonists as a potential therapy/prophylaxis for sickle cell complications has not been evaluated.
We found that patients with Hemoglobin SS (n=21) have abnormally elevated aldosterone plasma levels, as measured with ELISA, that range from 1.5–40 times (median: 8.6 times) higher than normal levels, similar in range to those of patients with heart failure (Struthers, Eur J of Heart Failure 2004). In addition, aldosterone levels in sickle cell patients positively correlated with secretory phospholipase A2 levels (R=0.43, p<0.05), a known biomarker for predicting acute chest syndrome. To determine how aldosterone affects endothelialsickle cell adhesion, we exposed human umbilical vein endothelial cells (HUVECs) and sickle erythrocytes and leukocytes isolated from patient samples to varying physiologic concentrations (1.0–100 nM) of aldosterone ex vivo for 2 hours and then utilized static and dynamic flow adhesion assays. We found that aldosterone increases sickle erythrocyte (but not normal erythrocytes), neutrophil and mononuclear cell (monocytes + lymphocytes) adhesion to endothelial cells in a dose-dependent manner (compared to controls, p<0.05 for all concentrations between 1–10 nM, p<0.001 for all concentrations >10nM) in static conditions. Compared to controls, endothelial-sickle blood cell adhesion increased up to 100 times with aldosterone exposure. Similarly, under physiologic flow conditions (shear stress: 1 dyne/cm2), endothelial cell exposure to aldosterone increased capture of sickle erythrocytes and leukocytes in a dose dependent manner (compared to controls, p<0.05 for all concentrations >10 nM). Furthermore, measurements with atomic force microscopy (AFM), a highly sensitive tool used to measure and track cell adhesion and deformability at the single cell level, revealed that the adhesive force between single sickle cell erythrocytes and HUVECs increases over time with aldosterone exposure. With the addition of spironolactone, an aldosterone antagonist, all adhesive interactions decreased to near baseline levels/controls (p>0.3 for all comparisons with baseline levels/controls) as measured with static and dynamic flow adhesion assays and AFM.
To investigate the underlying mechanisms of these phenomena, fluorescence imaging revealed increased reactive oxygen species production and expression of VCAM-1 and ICAM-1 in HUVECs exposed to aldosterone for only 2 hr when compared to controls. Aldosterone exposure did not affect sickle erythrocyte or leukocyte deformability as measured with ektacytometry and AFM, respectively. Taken together, these results suggest that aldosterone may play an important role in sickle cell vasculopathy and the high levels of this hormone may provide an effective therapeutic target for this disease.
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