James B. Parkerson

Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Differential action of angiotensin II and activity of angiotensin-converting enzyme in human bypass grafts

OBJECTIVE The activity of the renin-angiotensin system may be important in determining the performance of coronary artery bypass grafts. We have examined the activity of tissue angiotensin-converting enzyme and the effects of angiotensin II in vessels used as bypass grafts. METHODS Organ bath studies were used to determine the vasoactive effect of angiotensin II. The activity of the angiotensin-converting enzyme was assessed by metabolism of a specific synthetic substrate. RESULTS The saphenous vein produced greater maximum responses to angiotensin II than did the internal thoracic artery. This response was not modified by inhibition of nitric oxide synthase, cyclooxygenase, or by an endothelin receptor antagonist in either vessel. Losartan, an AT1 receptor antagonist, inhibited the vasoconstrictor response in both blood vessels. Homogenates of saphenous vein and internal thoracic artery displayed tissue angiotensin-converting enzyme activity, which was inhibited by captopril. Enzyme activity was threefold greater in the vein. Both the contractile response to angiotensin II and the enzyme activity were retained in venous grafts removed up to 20 years after coronary bypass surgery. CONCLUSIONS These data demonstrate that marked differences exist in angiotensin-converting enzyme activity and AT1 receptor responses in the saphenous vein compared with the internal thoracic artery. These findings may have important implications for the performance of the vein when used as a coronary artery bypass graft and may have clinical implications for the use of angiotensin-converting inhibitors and AT1 receptor antagonists in the prevention and treatment of vein graft disease.

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.

β-HYDROXYBUTYRATE INDUCES ACUTE PULMONARY ENDOTHELIAL DYSFUNCTION IN RABBITS

The authors examined the effects of high ketone body and glucose concentrations on endothelial cell (EC) function in perfused rabbit lungs. β-Hydroxybutyrate (βOHB), at 5 mM, decreased endothelial angiotensin-converting enzyme (eACE) activity, whereas 25 mM glucose (HG), 1 mM βOHB, or 10 mM acetoacetate (AcAc) did not. Dry to wet weight ratios were also reduced in lungs perfused with 5 mM βOHB, but not with AcAc. βOHB, at 5 mM, caused massive hemorrhage and interstitial and alveolar neutrophil infiltration; AcAc only produced engorgement of septal capillaries. Thus, pulmonary EC dysfunction occurs in rabbit lungs acutely perfused with βOHB, but not with AcAc or glucose.

Inhibition of pulmonary endothelial angiotensin converting enzyme activity by trandolaprilat in vivo

The purpose of the present study was to contrast a commonly used ACE inhibitor (enalaprilat) with a novel ACE inhibitor (trandolaprilat) in their ability to inhibit 1) pulmonary capillary endothelial‐bound ACE activity in vivo, 2) arterial pressure responses to i.v. angiotensin I and bradykinin, and 3) selected tissue ACE activity ex vivo, in rabbits. Pulmonary capillary endothelium‐bound ACE activity in vivo was estimated via the single pass transpulmonary hydrolysis of the substrate 3H‐Benzoyl‐Phenylalanyl‐Alanyl‐Proline (BPAP). Doses of acutely administered trandolaprilat (8 μg/kg) or enalaprilat (10 μg/kg) were equieffective in reducing the pressor response to angiotensin I. At these doses, trandolaprilat produced a greater inhibition of pulmonary capillary endothelial‐bound ACE activity (66.5 ± 4.7% reduction of baseline BPAP metabolism vs. 52.7 ± 4.2% by enalaprilat, P < 0.05). Chronically administered trandolaprilat (8 μkg/day for 8 days) was more effective than enalaprilat (either 8 μg/kg/day or 10 μg/kg/day for 8 days) in reducing the angiotensin‐1 induced increase in mean arterial pressure (increases of 9.7 ± 1.4 mmHg vs. 20.3 ± 2.3 mmHg and 19.1 ± 5.7 mmHg respectively; P < 0.01), as well as in reducing BPAP metabolism. In agreement with in vivo data, trandolaprilat was 5.5‐, 3.6‐, and 2.5‐times more effective than enalaprilat in reducing ACE activities in the aorta, left ventricle, and lung, respectively. We conclude that trandolaprilat is a more potent, longer acting, and more tissue‐selective ACE inhibitor than enalaprilat, and that the method outlined here can be used to aid in the development of tissue‐specific ACE inhibitors. Drug Dev. Res. 41:22–30, 1997.

Estimation of the dissociation constants for pulmonary endothelial angiotensin converting enzyme reactions with trandolaprilat and enalaprilat in vivo

We estimated the activity of pulmonary capillary endothelium‐bound (PCEB) angiotensin converting enzyme (ACE) in the rabbit in vivo, before and at 20 min and 2 h postadministration of the ACE inhibitors trandolaprilat (8 μg/kg) and enalaprilat (10 μg/kg), alone and in combination with the calcium channel blocker verapamil (100 μg/kg). PCEB ACE activity was assessed from the single‐pass transpulmonary hydrolysis of the synthetic substrate 3H‐benzoyl‐Phe‐Ala‐Pro (BPAP). We then calculated the modified kinetic parameter Amax/Km and the dissociation constants (k‐1) of the two inhibitors from PCEB ACE. Trandolaprilat reduced PCEB ACE activity less than enalaprilat, but its action was more sustained. Verapamil did not influence the immediate inhibitory action of either inhibitor. Enalaprilat exhibited more than a threefold higher k‐1 than trandolaprilat from PCEB ACE (77.9 ± 9.2 vs. 25.2 ± 4.3 × 10–5&sol/sec). Co‐injection of verapamil did not significantly affect the k‐1 of enalaprilat (86.3 ± 5.2 × 10–5/sec) but moderately increased that of trandolaprilat (45.2 ± 6 × 10–5/sec). We conclude that 1) trandolaprilat confers a longer‐lasting enzyme inhibition than enalaprilat, and 2) although the trandolaprilat‐verapamil treatment moderately reduces the duration of the trandolaprilat‐induced inhibition, it still offers a longer enzyme inhibition than enalaprilat alone or in combination with verapamil. Drug Dev. Res. 44:80–86, 1998.

Effect of nitrite on endothelial function in isolated lung.

Nitrated tyrosine, implicated in protein dysfunction, is increased in various tissues in association with diverse pathological processes. Angiotensin converting enzyme (ACE) is a luminal vascular endothelial enzyme whose dysfunction is an early sign of endothelial injury. ACE contains a tyrosine critical for its enzymatic activity. Others have shown that nitrite exacerbates the ACE dysfunction of cultured endothelial cells in contact with activated polymorphonuclear neutrophils (PMN). We hypothesized that exogenous nitrite would enhance endothelial ACE dysfunction associated with PMN activation in the isolated lung. Rats received lipopolysaccharide (LPS) 2 h prior to isolated lung perfusion with Ficoll containing buffer. Either formyl-Met-Leu-Phe (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 10(-7) M) was used to activate PMN in lungs treated or not treated with 300-microM nitrite. A first pass indicator dilution method and first order reaction kinetics were used to determine ACE activity, while lung Ficoll content served as an index of vascular permeability. Both fMLP and PMA decreased endothelial ACE activity and increased pulmonary artery pressure, edema and vascular permeability. Exogenous nitrate did not potentiate the decrease in ACE activity, the lung injury or nitrotyrosine immunoreactivity of lung homogenates. In contrast to observations in cultured endothelial cells, our findings in the whole lung are compatible with the speculation of others that the rat lung has an unidentified factor, which minimizes accumulation of nitrated proteins.