Clinical Hemorheology and Microcirculation 59 (2015) 67–81 DOI 10.3233/CH-141818 IOS Press
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Signature biomarkers in Diabetes Mellitus and associated Cardiovascular diseases Tanusri Karmakara , Sanjaya K. Mallickb , Arpita Chakrabortya , Animesh Maitic , Subhankar Chowdhuryd and Maitree Bhattacharyyaa,∗ a
Department of Biochemistry, and Center for Research in Nanoscience and Nanotechnology, University of Calcutta, Kolkata, India b BD-CU Center of Excellence for Nanobiotechnology, Kolkata, India c Medical College, Kolkata, India d IPGMER, Kolkata, India Abstract. Platelet signatures indicating differential dysfunction, hyperactivation, aggregation or adhesion are capable of expressing their characters during the journey of a disease process, and can be utilized as cost effective biomarkers with immense clinical value. Type 2 diabetes mellitus (T2DM) is a major lifestyle disease of contemporary world with progression to diabetes associated cardiovascular diseases (DM-CVD). We identified a few potential biomarkers in platelets of T2DM to analyze the thrombotic risk in diabetes subjects by utilizing flow cytometric quantification with different flurochrome conjugated monoclonal antibodies. Our study describes interesting correlations (p < 0.0001) for different clinical parameters of concurrent threat for vessel occlusion and the status of indices like reactive oxygen species, von Willebrand factor and mitochondrial membrane potential using western blotting and fluorescence techniques. Our study involved 32 T2DM, and 31 T2DM - CVD subjects compared to 29 healthy controls without any history of T2DM or CVD. An altered expression of platelet surface markers P-selectin (CD62p) and GpIIb/IIIa (CD 41/61, PAC1) along with changes in the platelet size due to agonist induced activation contributed to the enhanced thrombotic potential in the patients. This work elucidates the prospect of platelet biomarkers as diagnostic tool to predict cardiovascular risk in DM subjects. Keywords: Platelets, ROS, mitochondrial membrane potential, flow cytometry, surface markers, vWF
1. Introduction Diabetes mellitus is a major global health challenge of recent times resulting from consumption of high caloric food and sedentary lifestyle in concerted action with the genetic predisposition. Type 2 diabetes also known as non insulin dependent diabetes mellitus (NIDDM) is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency [18]. This rapidly growing disease, induces oxidative stress generating free radicals to disrupt normal cell signaling pathways [17, 23, 39]. Free radical generation again causes mitochondrial membrane depolarization to activate the apoptotic pathway. Moreover hyperglycemia in T2DM disrupts normal cellular homeostasis leading to the incidence of macrovascular complications with high rate of cardiovascular diseases like ischemic heart attack, atherosclerosis, stroke, angina, and coronary artery disease [24]. The pathogenesis of vascular diseases in diabetes is associated with dysfunction of blood platelets [37]. Platelet-mediated thrombosis includes different phases like adhesion, activation and aggregation/cohesion with clot formation [9]. Each state of platelet plug formation requires a coordinated response to ligand and receptor ∗
Corresponding author: Dr. Maitree Bhattacharyya, Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India. Tel.: +91 33 24614712 439; Fax: +91 33 24614849; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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T. Karmakar et al. / Signature biomarkers in diabetes mellitus and associated cardiovascular diseases
interactions, signaling molecules, membrane expression of clotting protein components, secretion of granule contents and cytoskeletal modifications. Abnormalities in any of these components results in platelet dysfunction which leads to anucleate cell fragments playing crucial role in managing vascular integrity and regulating hemostasis. Vascular occlusion by arterial thrombosis most often occurs after a rupture of an unstable atherosclerotic plaque [29]. Diabetes mellitus is a hypercoagulable state [5] and hyperglycemia plays an independent role in blood coagulation cascade directed to prothrombic state in T2DM [32]. Recently the independent prothrombotic state has been recognized as a component of the metabolic syndrome and subjects exhibit specific pattern of coagulation factors that promote thrombosis or retard thrombolysis [12]. The prothrombotic state is characterized by the expression of GlycoproteinIIb/IIIa (CD41/61and PAC1) and p-selectin (CD62p) mediating binding of von Willebrand factor and fibrin interactions which cause different abnormalities in platelet function. Several flurochrome conjugated monoclonal antibodies(MoAbs) recognize antigenic determinants on platelet surface after their activation detected with CD markers though platelet clinical research had to face problems so long due to their vulnerable nature, but recent advancement in flow cytometry inputs cutting edge technology to understand the functional stability of platelets and their surface receptors including P-selectin, CD41/61 and PAC1 in diseases like diabetes, renal diseases, Alzheimer’s and CVD.CD62p is the largest glycoprotein of the selectin family, with a mass of 140 kDa existing in the Weibel-Palade bodies of endothelial cells and the ␣-granules of platelets, playing a key role in platelet activation during the inflammatory process [3]. Surface receptor complex CD41/61 and PAC1 are members of a family of heterodimeric adhesive protein receptors and the cytoadhesion is expressed in type 2 diabetes with platelet hyperactivity. The interplay of hemorheological factors, oxidative stress and inflammation has a detrimental effect in diabetes due to the gross disturbance in microcirculation [13]. Platelets upon activation engage themselves in dynamic interaction with other blood cells mainly Red blood cells (RBCs) that affects platelet aggregation involved in primary hemostasis. ADP, the central agonist regulates platelet activation further leading to aggregation during hemostasis and thrombosis that operates through the G protein coupled receptors P2Y1 and P2Y12 [14]. Similarly Collagen, another agonist induce platelet activation by adhering to its specific surface receptor involved in integrin glycoprotein Ia/IIa [19, 26, 31]. In this study, we investigated expression of different platelet surface molecules along with ROS generation, mitochondrial membrane potential, vWF estimation and platelet size estimation to address the potential role of platelets involved in the association of DM and CVD. Until now most studies on platelet hemostatic function are limited to the western world context and almost negligible amount of work has been reported in Indian population. Although working with platelets is associated with certain experimental limitations, but we have exploited Flow cytometry (FACS), Spectrofluorometry, High Content Imaging Fluorescence (HCIF) microscope and Western blotting techniques to explore the status of platelet activity in T2DM and associated CVD. Hence, this work provides a unique attempt to correlate clinical parameters with platelet markers in diabetes associated cardiovascular complications.
2. Materials and methods 2.1. Study population A randomized study was performed among type 2 diabetes patients, samples being collected from Medical College, as well as IPGMER, SSKM Hospital, Kolkata, India. The study involved a total of 63
T. Karmakar et al. / Signature biomarkers in diabetes mellitus and associated cardiovascular diseases
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patients out of which 32 T2DM and 31 T2DM with CVD were involved for clinical trials compared to 29 controls without any history of T2DM or CVD. An informed consent for participation in the study was obtained from all diabetic and control individuals participating in this research program approved by Calcutta University Biosafety and Ethics Committee. All subjects were subdivided into three groups, the first group was control, second were T2DM with selection criteria including age group of 35–72 yrs, BMI of 23–34 kg/m2 , Glycosylated HbA1c (%) >6.0 and third were T2DM with CVD patients detected with either Ischemic Heart Diseases (IHD) or Stroke along with risk factors like hypertension (systolic pressure >130 mm Hg), hyperlipidemia (Total cholesterol >240,Triglyceride >200 LDL >135, HDL
67
Signature biomarkers in Diabetes Mellitus and associated Cardiovascular diseases Tanusri Karmakara , Sanjaya K. Mallickb , Arpita Chakrabortya , Animesh Maitic , Subhankar Chowdhuryd and Maitree Bhattacharyyaa,∗ a
Department of Biochemistry, and Center for Research in Nanoscience and Nanotechnology, University of Calcutta, Kolkata, India b BD-CU Center of Excellence for Nanobiotechnology, Kolkata, India c Medical College, Kolkata, India d IPGMER, Kolkata, India Abstract. Platelet signatures indicating differential dysfunction, hyperactivation, aggregation or adhesion are capable of expressing their characters during the journey of a disease process, and can be utilized as cost effective biomarkers with immense clinical value. Type 2 diabetes mellitus (T2DM) is a major lifestyle disease of contemporary world with progression to diabetes associated cardiovascular diseases (DM-CVD). We identified a few potential biomarkers in platelets of T2DM to analyze the thrombotic risk in diabetes subjects by utilizing flow cytometric quantification with different flurochrome conjugated monoclonal antibodies. Our study describes interesting correlations (p < 0.0001) for different clinical parameters of concurrent threat for vessel occlusion and the status of indices like reactive oxygen species, von Willebrand factor and mitochondrial membrane potential using western blotting and fluorescence techniques. Our study involved 32 T2DM, and 31 T2DM - CVD subjects compared to 29 healthy controls without any history of T2DM or CVD. An altered expression of platelet surface markers P-selectin (CD62p) and GpIIb/IIIa (CD 41/61, PAC1) along with changes in the platelet size due to agonist induced activation contributed to the enhanced thrombotic potential in the patients. This work elucidates the prospect of platelet biomarkers as diagnostic tool to predict cardiovascular risk in DM subjects. Keywords: Platelets, ROS, mitochondrial membrane potential, flow cytometry, surface markers, vWF
1. Introduction Diabetes mellitus is a major global health challenge of recent times resulting from consumption of high caloric food and sedentary lifestyle in concerted action with the genetic predisposition. Type 2 diabetes also known as non insulin dependent diabetes mellitus (NIDDM) is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency [18]. This rapidly growing disease, induces oxidative stress generating free radicals to disrupt normal cell signaling pathways [17, 23, 39]. Free radical generation again causes mitochondrial membrane depolarization to activate the apoptotic pathway. Moreover hyperglycemia in T2DM disrupts normal cellular homeostasis leading to the incidence of macrovascular complications with high rate of cardiovascular diseases like ischemic heart attack, atherosclerosis, stroke, angina, and coronary artery disease [24]. The pathogenesis of vascular diseases in diabetes is associated with dysfunction of blood platelets [37]. Platelet-mediated thrombosis includes different phases like adhesion, activation and aggregation/cohesion with clot formation [9]. Each state of platelet plug formation requires a coordinated response to ligand and receptor ∗
Corresponding author: Dr. Maitree Bhattacharyya, Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India. Tel.: +91 33 24614712 439; Fax: +91 33 24614849; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
68
T. Karmakar et al. / Signature biomarkers in diabetes mellitus and associated cardiovascular diseases
interactions, signaling molecules, membrane expression of clotting protein components, secretion of granule contents and cytoskeletal modifications. Abnormalities in any of these components results in platelet dysfunction which leads to anucleate cell fragments playing crucial role in managing vascular integrity and regulating hemostasis. Vascular occlusion by arterial thrombosis most often occurs after a rupture of an unstable atherosclerotic plaque [29]. Diabetes mellitus is a hypercoagulable state [5] and hyperglycemia plays an independent role in blood coagulation cascade directed to prothrombic state in T2DM [32]. Recently the independent prothrombotic state has been recognized as a component of the metabolic syndrome and subjects exhibit specific pattern of coagulation factors that promote thrombosis or retard thrombolysis [12]. The prothrombotic state is characterized by the expression of GlycoproteinIIb/IIIa (CD41/61and PAC1) and p-selectin (CD62p) mediating binding of von Willebrand factor and fibrin interactions which cause different abnormalities in platelet function. Several flurochrome conjugated monoclonal antibodies(MoAbs) recognize antigenic determinants on platelet surface after their activation detected with CD markers though platelet clinical research had to face problems so long due to their vulnerable nature, but recent advancement in flow cytometry inputs cutting edge technology to understand the functional stability of platelets and their surface receptors including P-selectin, CD41/61 and PAC1 in diseases like diabetes, renal diseases, Alzheimer’s and CVD.CD62p is the largest glycoprotein of the selectin family, with a mass of 140 kDa existing in the Weibel-Palade bodies of endothelial cells and the ␣-granules of platelets, playing a key role in platelet activation during the inflammatory process [3]. Surface receptor complex CD41/61 and PAC1 are members of a family of heterodimeric adhesive protein receptors and the cytoadhesion is expressed in type 2 diabetes with platelet hyperactivity. The interplay of hemorheological factors, oxidative stress and inflammation has a detrimental effect in diabetes due to the gross disturbance in microcirculation [13]. Platelets upon activation engage themselves in dynamic interaction with other blood cells mainly Red blood cells (RBCs) that affects platelet aggregation involved in primary hemostasis. ADP, the central agonist regulates platelet activation further leading to aggregation during hemostasis and thrombosis that operates through the G protein coupled receptors P2Y1 and P2Y12 [14]. Similarly Collagen, another agonist induce platelet activation by adhering to its specific surface receptor involved in integrin glycoprotein Ia/IIa [19, 26, 31]. In this study, we investigated expression of different platelet surface molecules along with ROS generation, mitochondrial membrane potential, vWF estimation and platelet size estimation to address the potential role of platelets involved in the association of DM and CVD. Until now most studies on platelet hemostatic function are limited to the western world context and almost negligible amount of work has been reported in Indian population. Although working with platelets is associated with certain experimental limitations, but we have exploited Flow cytometry (FACS), Spectrofluorometry, High Content Imaging Fluorescence (HCIF) microscope and Western blotting techniques to explore the status of platelet activity in T2DM and associated CVD. Hence, this work provides a unique attempt to correlate clinical parameters with platelet markers in diabetes associated cardiovascular complications.
2. Materials and methods 2.1. Study population A randomized study was performed among type 2 diabetes patients, samples being collected from Medical College, as well as IPGMER, SSKM Hospital, Kolkata, India. The study involved a total of 63
T. Karmakar et al. / Signature biomarkers in diabetes mellitus and associated cardiovascular diseases
69
patients out of which 32 T2DM and 31 T2DM with CVD were involved for clinical trials compared to 29 controls without any history of T2DM or CVD. An informed consent for participation in the study was obtained from all diabetic and control individuals participating in this research program approved by Calcutta University Biosafety and Ethics Committee. All subjects were subdivided into three groups, the first group was control, second were T2DM with selection criteria including age group of 35–72 yrs, BMI of 23–34 kg/m2 , Glycosylated HbA1c (%) >6.0 and third were T2DM with CVD patients detected with either Ischemic Heart Diseases (IHD) or Stroke along with risk factors like hypertension (systolic pressure >130 mm Hg), hyperlipidemia (Total cholesterol >240,Triglyceride >200 LDL >135, HDL
