Integration of VEGF and a-SMA Expression Improves the Prediction Accuracy of Fibrosis in Chronic Hepatitis C Liver Biopsy Shaimaa Elzamly, MD,* Hala A. Agina, MD,* Abd El-Latif Elbalshy, MD,* Maha Abuhashim, MD,w Eman Saad, MD,* and Zakaria Y. Abd Elmageed, PhDzy Introduction: The progression of fibrosis in chronic hepatitis C (CHC) is a multifactorial process. The high adverse effects and the cost of standard health care increase the demand to discover new predictors for the progression of fibrosis in CHC patients. Our study aims to establish the relation between the angiogenic marker [vascular endothelial growth factor (VEGF)] and acti- vated hepatic stellate cells (HSCs) represented by the expression of a-smooth muscle actin (a-SMA) and whether these 2 markers can be used as predictors for the progression of fibrosis in pa- tients with CHC. Materials and Methods: Histopathologic and immunohis- tochemical analyses were used for examining the morphology and the expression of VEGF and a-SMA in 60 CHC biopsies procured from CHC patients. Multivariate analysis was used to correlate the protein expression with staging and grading of liver fibrosis. Cutoff values of a-SMA and VEGF were determined by the receiver operating characteristics curve. Results: There was a positive correlation between VEGF and HSCs expressing a-SMA (r = 0.287, P = 0.026) and both fac- tors were correlated with the stage of fibrosis (P < 0.001). Using the receiver operating characteristics curve, both VEGF (area under the curve = 0.71, P < 0.006) and a-SMA (area under the curve = 0.82, P < 0.001) were positive predictors for moderate and severe fibrosis. Conclusions: This study demonstrates the relation between VEGF expression and the activated HSCs denoted by the ex- pression of a-SMA in CHC biopsies and together can be used as a predictor for the progression of fibrosis. Key Words: liver biopsy, fibrosis, chronic hepatitis C, VEGF, HSCs (Appl Immunohistochem Mol Morphol 2016;00:000–000) C hronic hepatitis C (CHC) is a progressive disease characterized by the development of hepatocellular necrosis, inflammation, and fibrosis. 1 Approximately 3% of the world population suffers CHC. 2 Liver biopsy re- mains the gold standard for the assessment of liver fib- rosis and is essential before the initiation of antiviral therapy in patients with CHC. 3,4 In patients with mild fibrosis, it is recommended to wait before starting ther- apy, whereas in moderate and severe fibrosis, a more aggressive approach is recommended. 5 Angiogenesis is the formation of new blood vessels from preexisting ones. 6 Normally, it occurs in different body organs as a response to injury and as an attempt of healing. Hepatic angiogenesis has been observed in the context of different inflammatory, fibrotic, and ischemic conditions. 7 It has been considered to be one of the fac- tors contributing to liver injury during CHC. 8,9 A rela- tionship between angiogenesis and hepatic fibrosis has long been suspected. 10 Experimental data have demon- strated that angiogenic factors expressed by hepatocytes had been implicated as a key event in the development of hepatic fibrosis through their interaction with the most potent fibrogenic cell: hepatic stellate cells (HSCs). 11,12 Among these factors, the vascular endothelial growth factor (VEGF) is the best characterized factor due to its mitogenic properties for endothelial cells. 6 VEGF belongs to a family made up by VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor. These factors bind to endothelial cell receptors with tyrosine kinase activity, such as Flt-1 and Flk-1. 11,13,14 The mesodermal origin of undifferentiated fetal HSC is supported by the fact that they express a-smooth Received for publication June 21, 2015; accepted October 14, 2015. From the *Pathology Department, Faculty of Medicine, Benha Uni- versity, Benha; wPathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt; Departments of zSurgery; and yOtolaryngology, Tulane University School of Medicine, New Orleans, LA. Z.Y.A.E. and H.A.A.: conceived the study, discussed the data, and wrote, reviewed, and edited the final version of the manuscript; S.E.: performed the experiments, analyzed the data, and wrote and re- viewed the final version of the manuscript; A.E.-L.E., M.A., E.S.: performed the experiments, provided the tissue specimens, discussed the results, and performed the statistical analysis. The authors declare no conflict of interest. Reprints: Zakaria Y. Abd Elmageed, PhD, and Shaimaa Elzamly, MD, Department of Surgery, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (e-mails: zabdelma@ tulane.edu, shaimaa.abdelfattah@fmed.bu.edu.eg). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.appliedimmunohist.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved. RESEARCH ARTICLE Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2016 www.appliedimmunohist.com | 1 Copyright r 2016 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.