Seojin Kang

Low level of lead can induce phosphatidylserine-exposure and erythrophagocytosis:A new mechanism underlying lead-associated anemia

Anemia is probably one of the most well-known toxic effects of lead. Previously, lead-induced anemia was considered to be from the inhibition of δ-aminolevulinic acid dehydratase participating in the heme biosynthesis. However, little is known whether lead could affect the destruction of erythrocyte, another important factor for anemia. In the present study, we demonstrated that lead could accelerate the splenic sequestration of erythrocytes through phosphatidylserine (PS) exposure and subsequently increased erythrophagocytosis. In freshly isolated human erythrocytes, Pb(2+)- induced PS exposure at relatively low concentrations (∼0.1 μM) by inhibiting flippase, a key aminophospholipid translocase for the maintenance of PS asymmetry and adenosine triphosphate depletion appeared to underlie this phenomenon. Abnormal shape changes of erythrocytes and microvesicle generation and other triggers for the erythrophagocytosis were also observed in the Pb(2+)-exposed erythrocytes. In vitro data showed that human macrophage indeed recognized and phagocytosis PS-exposed erythrocytes. In good accordance with these in vitro results, the oral administration of Pb(2+) increased PS exposure on erythrocytes in rat in vivo. In addition, reduction of hematocrit and hemoglobin and increased spleen weight were observed along with enhanced splenic sequestration of erythrocytes in the rats exposed to Pb(2+) subchronically for 4 weeks through drinking water. In conclusion, these results suggest that Pb(2+)-induced anemia may be explained at least in part by increased PS exposure on erythrocytes, erythrophagocytosis, and splenic sequestration.

Doxorubicin-induced platelet procoagulant activities: an important clue for chemotherapy-associated thrombosis.

Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported; yet, the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle (MP) generation. Consistently, DOX-treated platelets and generated MPs induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca²⁺ increase and ATP depletion, which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Δψ), cytochrome c release, Bax translocation, and caspase-3 activation. A Ca²⁺ chelator ethylene glycol tetraacetic acid, caspase inhibitor Q-VD-OPh, and antioxidants (vitamin C and trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca²⁺, apoptosis, and reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity, which was accompanied by increased PS exposure and Δψ dissipation in platelets.

Novel Antiplatelet Activity of Protocatechuic Acid through the Inhibition of High Shear Stress-Induced Platelet Aggregation

Bleeding is the most common and serious adverse effect of currently available antiplatelet drugs. Many efforts are being made to develop novel antithrombotic agents without bleeding risks. Shear stress-induced platelet aggregation (SIPA), which occurs under abnormally high shear stress, plays a crucial role in the development of arterial thrombotic diseases. Here, we demonstrate that protocatechuic acid (PCA), a bioactive phytochemical from Lonicera (honeysuckle) flowers, selectively and potently inhibits high shear (>10,000 s−1)-induced platelet aggregation. In isolated human platelets, PCA decreased SIPA and attenuated accompanying platelet activation, including intracellular calcium mobilization, granule secretion, and adhesion receptor expression. The anti-SIPA effect of PCA was mediated through blockade of von Willebrand factor binding to activated glycoprotein Ib, a primary and initial event for the accomplishment of SIPA. Conspicuously, PCA did not inhibit platelet aggregation induced by other endogenous agonists like collagen, thrombin, or ADP that are important in both pathological thrombosis and normal hemostasis. Antithrombotic effects of PCA were confirmed in vivo in a rat arterial thrombosis model, where PCA significantly delayed the arterial occlusion induced by FeCl3. Of particular note, PCA did not increase bleeding times in a rat tail transection model, whereas conventional antiplatelet drugs, aspirin, and clopidogrel substantially prolonged it. Collectively, these results suggest that PCA may be a novel antiplatelet agent that can prevent thrombosis without increasing bleeding risks.

Erythrophagocytosis of Lead-Exposed Erythrocytes by Renal Tubular Cells: Possible Role in Lead-Induced Nephrotoxicity

Background: Nephrotoxicity associated with lead poisoning has been frequently reported in epidemiological studies, but the underlying mechanisms have not been fully described. Objectives: We examined the role of erythrocytes, one of the major lead reservoirs, in lead-associated nephrotoxicity. Methods and results: Co-incubation of lead-exposed human erythrocytes with HK-2 human renal proximal tubular cells resulted in renal tubular cytotoxicity, suggesting a role of erythrocytes in lead-induced nephrotoxicity. Morphological and flow cytometric analyses revealed that HK-2 cells actively phagocytized lead-exposed erythrocytes, which was associated with phosphatidylserine (PS) externalization on the erythrocyte membrane and generation of PS-bearing microvesicles. Increased oxidative stress and up-regulation of nephrotoxic biomarkers, such as NGAL, were observed in HK-2 cells undergoing erythrophagocytosis. Moreover, TGF-β, a marker of fibrosis, was also significantly up-regulated. We examined the significance of erythrophagocytosis in lead-induced nephrotoxicity in rats exposed to lead via drinking water for 12 weeks. We observed iron deposition and generation of oxidative stress in renal tissues of lead-exposed rats, as well as the histopathological alterations such as tubulointerstitial lesions, fibrosis, and up-regulation of KIM-1, NGAL, and TGF-β. Conclusions: Our data strongly suggest that erythrophagocytosis and subsequent iron deposition in renal tubular cells could significantly enhance nephrotoxicity following lead exposure, providing insight on lead-associated kidney damages. Citation: Kwon SY, Bae ON, Noh JY, Kim K, Kang S, Shin YJ, Lim KM, Chung JH. 2015. Erythrophagocytosis of lead-exposed erythrocytes by renal tubular cells: possible role in lead-induced nephrotoxicity. Environ Health Perspect 123:120–127; http://dx.doi.org/10.1289/ehp.1408094

Potentiation of vasoconstriction and pressor response by low concentration of monomethylarsonous acid (MMAIII)

A close link between arsenic exposure and hypertension has been well-established through many epidemiological reports, yet the mechanism underlying it remains unclear. Here we report that nanomolar concentrations of monomethylarsonous acid (MMA(III)), a toxic trivalent methylated arsenic metabolite, can potentiate agonist-induced vasoconstriction and pressor responses. In freshly isolated rat aortic ring, exposure to nanomolar MMA(III) (100-500 nM) potentiated phenylephrine (PE)-induced vasoconstriction while at higher concentrations (≥2.5 μM), suppression of vasoconstriction and apoptosis of vascular smooth muscle were observed. Potentiation of agonist-induced vasoconstriction was also observed with other contractile agonists and it was retained in endothelium-denuded aortic rings, suggesting that these events are agonist-independent and smooth muscle cell dependent. Interestingly, exposure to MMA(III) resulted in increased myosin light chain phosphorylation while PE-induced Ca2+ influx was not affected, reflecting that Ca2+ sensitization is involved. In line with this, MMA(III) enhanced agonist-induced activation of small GTPase RhoA, a key contributor to Ca2+ sensitization. Of note, treatment of MMA(III) to rats induced significantly higher pressor responses in vivo, demonstrating that this event can occur in vivo indeed. We believe that RhoA-mediated Ca2+ sensitization and the resultant potentiation of vasoconstriction by MMA(III) may shed light on arsenic-associated hypertension.

Inhibitory effects of black soybean on platelet activation mediated through its active component of adenosine

Owing to the beneficial health effects on human cardiovascular system, soybeans and soy-related products have been a focus of intensive research. Soy isoflavones are known to be primarily responsible for the soy-related biological effects including anti-platelet activity but its in vivo relevancy has not been fully verified. Here we compared the role of adenosine, an active ingredient abundant in black soybean (BB) extract, in the anti-platelet effects of BB, to that of soy isoflavones. At the concentrations existing in BB, isoflavones such as genistein and daidzein could not attenuate collagen-induced platelet aggregation, however, adenosine significantly inhibited platelet aggregation with an equivalent potency to BB, suggesting that adenosine may be the major bioactive component. Consistently, the anti-aggregatory effects of BB disappeared after treatment of adenosine receptor antagonists. The effects of BB are mediated by adenosine through intracellular cAMP and subsequent attenuation of calcium mobilization. Of note, adenosine and BB significantly reduced platelet fibrinogen binding and platelet adhesion, other critical events for platelet activation, which were not affected by isoflavones. Taken together, we demonstrated that adenosine might be the major active ingredient for BB-induced anti-platelet activity, which will shed new light on the roles of adenosine as a bioactive compound in soybeans and soy-related food.

Emodin inhibits tonic tension through suppressing PKCδ-mediated inhibition of myosin phosphatase in rat isolated thoracic aorta

Dysregulated tonic tension and calcium sensitization in blood vessels has frequently been observed in many cardiovascular diseases. Despite a huge therapeutic potential, little is known about natural products targeting tonic tension and calcium sensitization.

High-Dose Vitamin C Injection to Cancer Patients May Promote Thrombosis Through Procoagulant Activation of Erythrocytes

Potential risk of high-dose vitamin C consumption is often ignored. Recently, gram-dose vitamin C is being intravenously injected for the treatment of cancer, which can expose circulating blood cells to extremely high concentrations of vitamin C. As well as platelets, red blood cells (RBCs) can actively participate in thrombosis through procoagulant activation. Here, we examined the procoagulant and prothrombotic risks associated with the intravenous injection of gram-dose vitamin C. Vitamin C (0.5-5 mM) increased procoagulant activity of freshly isolated human RBCs via the externalization of phosphatidylserine (PS) to outer cellular membrane and the formation of PS-bearing microvesicles. PS exposure was induced by the dysregulation of key enzymes for the maintenance of membrane phospholipid asymmetry, which was from vitamin C-induced oxidative stress, and resultant disruption of calcium and thiol homeostasis. Indeed, the intravenous injection of vitamin C (0.5-1.0 g/kg) in rats in vivo significantly increased thrombosis. Notably, the prothrombotic effects of vitamin C were more prominent in RBCs isolated from cancer patients, who are at increased risks of thrombotic events. Vitamin C-induced procoagulant and prothrombotic activation of RBCs, and increased thrombosis in vivo. RBCs from cancer patients exhibited increased sensitivity to the prothrombotic effects of vitamin C, reflecting that intravenous gram-dose vitamin C therapy needs to be carefully revisited.

Dysfunction of Vascular Smooth Muscle and Vascular Remodeling by Simvastatin

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in time- and concentration-dependent manners (0.86 g ± 0.11 at 10 μM simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca²⁺ channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca²⁺ increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonist-induced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca²⁺ influx and RhoA activity, ultimately leading to apoptosis and structural remodeling.

Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity

Statins, lipid-lowering agents for the prevention of atherosclerosis and fatal coronary heart diseases, have pleiotropic modalities on the function and physiology of vascular smooth muscle that include anti-contractile and pro-apoptotic effects. These effects were suggested to stem from the inhibition of small GTPase Rho A, but they are largely regarded as distinct and unrelated. Recently, we discovered that simvastatin causes both contractile dysfunction and apoptosis of vascular smooth muscle cells (VSMCs), reflecting that they may be closely related, yet their connecting link remains unexplained. Here, we elaborated the mechanism underlying simvastatin-induced apoptosis of normal VSMCs in connection with contractile dysfunction. Repeated oral administration of simvastatin to rats in vivo resulted in contractile dysfunction and apoptosis of vascular smooth muscle, of which pattern was well reproduced in rat VSMCs in vitro. Of note, contractile dysfunction and apoptosis occurred in concerted manners both in vivo and in vitro in the aspects of time course and dose of exposure. In rat VSMCs, simvastatin impaired the activation of small GTPases, RhoA along with Rac-1, which resulted in the disruption of actin integrity, a pivotal factor both for the generation of contractile force and survival of VSMCs. In line with the disruption of actin integrity, Bmf, a pro-apoptotic factor bound to intact actin, dissociated and translocated into mitochondria, which corresponded well with the dissipation of mitochondrial membrane potential, caspase-3 activation and ultimately apoptosis. These events were all rescued by an actin stabilisation agent, jasplakinolide as well as geranylgeraniol, indicating that damages of the actin integrity from disrupted activation of RhoA/Rac-1 lies at the center of simvastatin-induced contractile dysfunction and apoptosis in vascular smooth muscle.