Indian Journal of Pathology and Microbiology
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Year : 2014  |  Volume : 57  |  Issue : 3  |  Page : 427-430
Phosphatase and tensin analog expression in arterial atherosclerotic lesions

1 Department of Pathology, Chennai Medical College, Trichy, Tamil Nadu, India
2 Department of Pathology, JIPMER, Puducherry, India
3 Department of Cardiothoracic and Vascular Surgery, JIPMER, Puducherry, India

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Date of Web Publication14-Aug-2014


Context: Phosphatase and tensin analog (PTEN) gene mutation has been proven for pro-inflammatory property and proliferative potential through tyrosine kinase pathway. We studied mutated PTEN for its pathogenetic association in arterial atherosclerosis. Aims: The objective was to study mutation of PTEN by immunohistochemical method in arterial atherosclerotic lesions and correlate with grades of atherosclerosis, smooth muscle migration in intima, degree of inflammation and Framingham heart study risk factors. Settings and Design: Human, Prospective Clinical study. Materials and Methods: We studied patients with arterial occlusive disease diagnosed by Doppler ultrasonography over a 2-year period. Immunohistochemistry was performed with mouse monoclonal antibodies for PTEN and smooth muscle actin (SMA). Statistical Analysis Used: Chi-square test. Results and Conclusion: Aorta was the single most common vessel affected (21%). Mean age of patients studied was 48.6 years and 80% were male. Mutant PTEN was associated with higher grades of atherosclerotic lesions (P < 0.0001) graded by American Heart Association classification and with smooth muscle proliferation and migration in intima (P < 0.0001). No statistically significant association with the vessel wall inflammation and other risk factors of atherosclerosis.

Keywords: Arterial occlusive disease, atherosclerosis, phosphatase and tensin analog

How to cite this article:
Muthalagan E, Ganesh RN, Sai Chandran B V, Verma SK. Phosphatase and tensin analog expression in arterial atherosclerotic lesions. Indian J Pathol Microbiol 2014;57:427-30

How to cite this URL:
Muthalagan E, Ganesh RN, Sai Chandran B V, Verma SK. Phosphatase and tensin analog expression in arterial atherosclerotic lesions. Indian J Pathol Microbiol [serial online] 2014 [cited 2021 Nov 27];57:427-30. Available from: https://www.ijpmonline.org/text.asp?2014/57/3/427/138741

   Introduction Top

Phosphatase and tensin analog (PTEN) is a tumor suppressor gene, expressed in endothelial cells, sub-endothelial cells and vascular smooth muscle cells. It regulates their proliferation, migration, survival/apoptosis, hypertrophy, and contractility. [1],[2],[3]

All patients with arterial occlusive disease diagnosed by Doppler ultrasonography from 2010 to 2012 were included in the study, while trauma and cardiac causes were excluded from the study by performing Echocardiogram (ECHO). We analyzed histo-pathology of the vessel wall biopsies at the site of thrombosis to study the role of PTEN mutation in various stages of atherosclerosis. Its expression was correlated with a degree of inflammation, smooth muscle proliferation in the intima and neo-vascularization. Mutant PTEN is associated with the absence of staining of PTEN antibody by immunohistochemistry. [4]

Patient's clinical profile, including age, gender, site of vessel biopsy, diabetic status, hypertension, history of smoking, alcoholism, and ECHO finding were documented. Hypertensive status of the patients was classified according to the Joint National Committee seven classification; [5] Diabetic status was classified as per WHO guidelines.

Other biochemical parameters such as a lipid profile and hemogram, platelet count, prothrombin time (international normalized ratio), activated partial thromboplastin time and thrombin time were collected. Patient's lipid profile was classified based on the recommendation from National Cholesterol Education program 2001.

Histopathological changes were studied with hematoxyiln and eosin (H and E), Verhoeff Van Gieson, and Masson Trichrome in the vessel walls. Histopathological changes like intimal macrophage accumulation, intracellular and extracellular lipid deposition, intimal/medial calcification, intimal fibrosis, smooth muscle proliferation and migration to intima, inflammatory cell infiltration, and neo-vascularization were studied in the biopsies. American Heart Association (AHA) Classification for Atherosclerosis was used to grade the lesions in the vessel wall biopsies.

Inflammation in atherosclerosis was semi-quantitatively scored based on the presence of inflammatory cells other than macrophages in H and E sections. Macrophages were excluded as they are quantified as part of AHA grading. The various grades were

  • Grade 0: no cells
  • Grade 1: 1-10 inflammatory cells/HPF
  • Grade 2: 10-20 inflammatory cells/HPF
  • Grade 3: >20 inflammatory cells/HPF.

Immunohistochemistry was performed with monoclonal antibodies for PTEN, SMA (smooth muscle actin) and CD31 (endothelial marker) using monoclonal mouse antibodies from DAKO antibodies USA by Envision technique.

Phosphatase and tensin analog normally stains the nucleus of endothelial cells and nucleus of tunica media. PTEN expression was studied for the presence or absence (mutation) of staining.

Phosphatase and tensin analog mutation was associated with the loss of expression. Absence and various degrees of expression were correlated with AHA grading, degree of inflammation and neo-vascularization and other risk factors.

CD31, a marker for endothelial cells was used for identifying and quantifying the intimal neo-vascularization. Neo-vascularization in thrombosed vessel was semi-quantitatively assessed as follows,

  • Grade 0 = No neo-vascularization
  • Grade I = 1-25% of intima
  • Grade II = 26-50% of intima
  • Grade III = >50% of intima.

SMA, smooth muscle actin stain was used to quantify the smooth muscle proliferation and migration to intima. Biopsies were grouped in to two categories based on the presence or the absence of the smooth muscle proliferation.

   Results Top

A total of 70 patients were studied. Of these, 14 patients were excluded as they showed only fibrin thrombi, lack of full thickness of vessels, and lack of complete details. One patient showed tumor emboli from pulmonary adenocarcinoma and two patients had emboli of cardiac origin proven by ECHO findings.

In our study, we observed that smokers contributed 52% while 37% were alcoholic and 56% had hypercholesterolemia. ECHO findings were normal in 63%, whereas 21% had left ventricular hypertrophy and the rest (16%) showed inferior wall hypokinesia, left ventricular dysfunction and severe aortic regurgitation.

Endothelial lining in tunica intima was preserved in 32 biopsies, while in the rest, there was ulceration and rupture of the endothelial lining and extensive damage to tunica media. Histopathological features were analyzed and subsequently IHC staining for PTEN, CD31, and SMA were performed in the 32 biopsies. Remaining 24 had extensive damage to tunica media and intima.

The 32 biopsies were classified according to AHA grading of Atherosclerosis. Class I AHA lesion had 1 patient, Class III 7, Class IV 12, Class V 5 and Class VI 7, while there was none in Class II. Duplication of internal elastic lamina was observed from Grade III AHA lesions upwards in higher grades [Figure 1]b. Chi-square test was used to identify the degree of correlation between PTEN mutation and smooth muscle proliferation and migration in intima by SMA staining, [Figure 1]c and significance of association was found to be (P < 0.0001) highly significant [Table 1].
Table 1: PTEN mutation and smooth muscle proliferation and migration

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Figure 1: (a) Normal endothelial cells and tunica media shows positi vity (positi ve control) (IHC-DAB stain, ×400), (b) duplicati on of internal elasti c lamina in Grade III lesion (VVG stain, ×100), (c) Sma highlights the smooth muscle proliferati on in Grade V lesion (IHC-DAB stain, ×40) (d) CD31 highlights the neovascularizati on in inti ma of Grade VI lesion (IHC-DAB stain ×40)

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Phosphatase and tensin analog mutation was correlated with AHA grade in two groups, early lesions (Group I) comprising AHA Grades I through IV, while advanced lesions (Group II) comprised of Class V and VI lesion [Figure 2]a-d. This grouping was done to facilitate statistical analysis as the total patients in each group were too few. The significance of association was found to be (P < 0.0001) highly significant by Chi-square test [Table 2].
Table 2: PTEN mutati on and AHA grade

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On applying one-way ANOVA for correlation between the degree of inflammation and neo-vascularization in tunica intima [Figure 1]d with PTEN expression, P = 0.09 [Table 3] and 0.1 [Table 4], respectively.
Table 3: PTEN mutation and score of intimal inflammation

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Table 4: PTEN mutation and intimal neovascularization

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Figure 2: (a) Histologic section from Grade IV atherosclerotic lesion (H and E stain, ×40) (b) Strong positivity for PTEN in Grade IV lesion (IHCDAB stain, ×400) (c) Histologic section from Grade VI atherosclerotic lesion (H and E stain, ×40), (d) Negativity of PTEN in Grade VI lesion (IHC-DAB stain, ×400)

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Age, smoking, alcoholism, diabetic status, hypertension, and hypercholesterolemia had no significant statistical association with PTEN mutation.

Mutation of PTEN was a localized phenomenon and it was seen only at the site of thrombosis.

   Discussion Top

The role of PTEN in smooth muscle proliferation, inflammation and neo-intimal formation has been highlighted by various authors in pathogenesis of coronary artery disease. [6],[7]

Of the 70 biopsies studied, we could study the detailed histo-pathology and immunohistochemistry in only 32 biopsies. Vessel wall biopsies of the 24 cases from site of arterial occlusion represented damaged vessel walls and in many of them tunica intima was not represented.

American Heart Association Grades V and VI lesions were seen in 12 patients only. In remaining 20 patients with manifestations of arterial occlusive disease, we found lesser AHA grades of atherosclerosis (Grades I-IV). In these patients, vasospasm is the most likely cause of arterial occlusion.

One patient suffered from Marfan syndrome and had manifestations of elastic fiber and cystic medial degeneration in the tunica media with ensuing aortic aneurysm. In seven patients, there was evidence of plaque rupture.

Our study did not find any significant association between the major clinical risk factors of atherosclerosis as exemplified by Framingham Heart study.

Over-expression of PTEN is reported to decrease smooth muscle proliferation through the direct effect on its growth and survival. [8],[9] This finding was strongly correlated in our study with very high statistical significance for PTEN mutation and increase in smooth muscle proliferation in tunica intima of occlusive arteries.

Mutation of PTEN was significantly associated with increasing grades of atherosclerosis as classified by AHA.

Our findings did not correlate with hypothesis of Koide S et al., that PTEN plays an essential role in endogenous function of cells in cardiovascular system with particular emphasis on neointimal formation and inflammatory cytokines. [10]

Brevetti et al. in their study found that associations between inflammatory factors and atherosclerosis may differ between the coronary and lower extremity arterial beds. [11],[12] Our study did not show association between PTEN mutation with degree of inflammation or neovascularisation in atherosclerosis.

   Conclusion Top

Our study assumes significance as it focuses on pathogenesis of peripheral arterial occlusion. Most available literature focuses on coronary arteries without histopathological evidence from living human subjects as they are from autopsies or experimental animals. Furthermore, pathologic features of coronary artery occlusion may be different from other arteries in body as coronaries differ from other arteries in following aspects: (i) Coronaries are the only arteries, which keeps moving and are externally compressed and released throughout (systoic compression within the myocardium) - almost 70-80 times/min. Coronaries have peculiar anatomy, these are the smallest artery (3-4 mm) directly originating from the largest artery (23-25 mm) while, other arteries originate after gradual tapering in diameter by arborisation pattern. These features make coronary arteries more stress-prone causing accelerated atherosclerosis.

   Acknowledgment Top

Technical Staff of Department of Pathology for whole hearted support.

   References Top

Vasudevan KM, Gurumurthy S, Rangnekar VM. Suppression of PTEN expression by NF-kappa B prevents apoptosis. Mol Cell Biol 2004;24:1007-21.  Back to cited text no. 1
Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, Backx PH. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J Mol Cell Cardiol 2004;37:449-71.  Back to cited text no. 2
Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T, et al. Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 2002;110:737-49.  Back to cited text no. 3
Kurose K, Zhou XP, Araki T, Cannistra SA, Maher ER, Eng C. Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am J Pathol 2001;158:2097-106.  Back to cited text no. 4
Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003;42:1206-52.  Back to cited text no. 5
Jia G, Mitra AK, Gangahar DM, Agrawal DK. Regulation of cell cycle entry by PTEN in smooth muscle cell proliferation of human coronary artery bypass conduits. J Cell Mol Med 2009;13:547-54.  Back to cited text no. 6
Selvendiran K, Kuppusamy ML, Bratasz A, Tong L, Rivera BK, Rink C, et al. Inhibition of vascular smooth-muscle cell proliferation and arterial restenosis by HO-3867, a novel synthetic curcuminoid, through up-regulation of PTEN expression. J Pharmacol Exp Ther 2009;329:959-66.  Back to cited text no. 7
Huang J, Niu XL, Pippen AM, Annex BH, Kontos CD. Adenovirus-mediated intraarterial delivery of PTEN inhibits neointimal hyperplasia. Arterioscler Thromb Vasc Biol 2005;25:354-8.  Back to cited text no. 8
Hata JA, Petrofski JA, Schroder JN, Williams ML, Timberlake SH, Pippen A, et al. Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts. J Thorac Cardiovasc Surg 2005;129:1405-13.  Back to cited text no. 9
Koide S, Okazaki M, Tamura M, Ozumi K, Takatsu H, Kamezaki F, et al. PTEN reduces cuff-induced neointima formation and proinflammatory cytokines. Am J Physiol Heart Circ Physiol 2007;292:H2824-31.  Back to cited text no. 10
Brevetti G, Giugliano G, Brevetti L, Hiatt WR. Inflammation in peripheral artery disease. Circulation 2010;122:1862-75.  Back to cited text no. 11
AbuRahma AF, Richmond BK, Robinson PA. Etiology of peripheral arterial thromboembolism in young patients. Am J Surg 1998;176:158-61.  Back to cited text no. 12

Correspondence Address:
Rajesh Nachiappa Ganesh
Department of Pathology, JIPMER, Puducherry
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Source of Support: Intramural Research Fund, JIPMER., Conflict of Interest: None

PMID: 25118736

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4]


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