Angiogenesis in Cutaneous Lesions of Leprosy: Implications for Treatment

STUDY Angiogenesis in Cutaneous Lesions of Leprosy Implications for Treatment

Sulochana S. Bhandarkar, MD; Cynthia Cohen, MD; Maria Kuruvila, MD; Thomas H. Rea, MD; Jamie B. MacKelfresh, MD; Delphine J. Lee, MD, PhD; Robert L. Modlin, MD; Jack L. Arbiser, MD, PhD

Objective: To examine the potential role of angiogen- Main Outcome Measure: CD31 microvessel counts. esis in leprosy. Results: The mean CD31 microvessel count in border- Design: Immunohistochemical analysis of leprosy lesions. line tuberculoid, midborderline, and lepromatous leprosy lesions was significantly higher than in indetermi- Setting: Department of Dermatology, Venereology, and nate leprosy lesions. Leprology, Kasturba Medical College; Division of Derma- tology, University of California at Los Angeles; and Depart- Conclusions: Increased bacterial load is associated with ments of Dermatology and Pathology, Emory University. increased angiogenesis. Angiogenesis inhibitors may be of benefit in the treatment of leprosy. Patients: Thirty-two cutaneous lesions that represented the spectrum of leprosy were obtained from 32 patients. Arch Dermatol. 2007;143(12):1527-1529

EPROSY IS A CHRONIC INFEC- ability of the host to mount an IL-12 re- tion caused by Mycobacte- sponse to the pathogen.7 IL-12 was shown rium leprae and is endemic to to be a potent angiogenesis inhibitor, as tropical areas of the world, in- were some of the cytokines induced by IL- cluding the Indian subconti- 12, including interferon-inducible pro- nent,L1 Indochina, and South America.2 Lep- tein 10 and interferon gamma.8-13 Thalido- rosy causes much morbidity through mide, a drug used to treat leprosy, also has formation of trophic ulcers, eye disabili- antiangiogenic properties.14 Minocy- ties, neuropathy, and social isolation. Al- cline, a drug that is a part of the rifampin, though leprosy is currently treatable, treat- ofloxacin, and minocycline regimen given ment of leprosy requires long courses of for single skin lesions such as paucibac- multiple antibiotics, which can decrease illary leprosy, has antiangiogenic proper- compliance.3 During treatment, patients can ties through inhibition of matrix metallo- experience debilitating erythema nodo- proteinases.15 Given the potential role of Author Affiliations: sum leprosum and reversal reactions.4 Fi- angiogenesis in leprosy and also since Departments of Dermatology nally, the irreversible neural damage and its M leprae, the causative organism of lep- (Drs Bhandarkar, MacKelfresh, associated sensory defects lead to the de- rosy, cannot be cultured, we wanted to and Arbiser) and Pathology formities and disabilities that form a part of study the vascularity of the entire spec- (Dr Cohen), Emory University 5 School of Medicine, Atlanta, the social stigma associated with leprosy. trum of leprosy through histologic assess- Georgia; Department of Leprosy exists in a clinical spectrum of ment. Our findings demonstrate an Microbiology, Immunology, and disease, ranging from solitary inflamma- increase in angiogenesis toward the lep- Molecular Genetics, University tory lesions called tuberculoid to dissemi- romatous spectrum lesions and raise the of California at Los Angeles nated disease characterized by formation possibility that angiogenesis inhibitors may (Drs Lee and Modlin); of highly bacilliferous granulomas, which be useful in the treatment of leprosy. Department of Dermatology, is termed lepromatous.6,7 Leprosy, like other Venereology, and Leprology, mycobacterial infections (tuberculosis), METHODS Kasturba Medical College, leishmaniasis, and syphilis, requires in- Mangalore, India 7 (Drs Bhandarkar and Kuruvila); tact cellular immunity for clearance. In- We studied 32 cutaneous lesions that repre- and Department of terleukin 12 (IL-12) has been demon- sented the spectrum of leprosy from 32 pa- Dermatology, University of strated to play a pivotal role in the tients for expression of CD31. Twenty-eight par- Southern California, clearance of these pathogens, and the clini- affin blocks were received from the Department Los Angeles (Dr Rea). cal spectrum of leprosy likely reflects the of Dermatology, Venereology, and Leprology,

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 Kasturba Medical College, and 4 sections were received from the for 15 minutes. The mean CD31 microvessel density was quan- Division of Dermatology, University of California at Los Ange- titated microscopically by 2 independent observers (C.C. and les. The diagnosis of leprosy in the patients was supported by he- J.B.M.). The number of CD31-positive blood vessels in the whole matoxylin-eosin–stained histologic analyses of the biopsied le- biopsy specimen and in 2 hot spots at a power of ϫ20 was de- sions and the clinical history of the patients. We studied 4 sections termined according to the method of Weidner et al.18 Hot spots of indeterminate leprosy, 5 of tuberculoid leprosy, 12 of border- were areas determined by the observers to represent the fields of line tuberculoid leprosy, 3 of midborderline leprosy, 3 of bor- greatest vascular density within a given section.19 The mean num- derline lepromatous leprosy, and 5 of lepromatous leprosy. ber of microvessels in 2 hot spots from each section was determined, and the total mean of each spectrum was compared with IMMUNOHISTOCHEMICAL ANALYSIS indeterminate leprosy.

A total of 32 sections (5 mm) of formalin-fixed, paraffin- STATISTICAL ANALYSIS embedded tissue (5 µm) were immunostained with monoclonal Ͻ antibodies against CD31 (1:80) (clone JC170A, 1/80; Dako Corp, P .05 was considered statistically significant. A t test was used Carpinteria, California) using a horseradish peroxidase–labeled for statistical analysis, which was performed with Excel (Mi- polymer, a heat-induced antigen retrieval, and an autostainer crosoft Inc, Redmond, Washington). (Dako).16,17 The Dako Envision system is a 2-step horseradish peroxidase–labeled polymer that is conjugated with secondary an- RESULTS tibodies and is used in combination with the automated Dako Au- tostainer. Hematoxylin was used as the counterstain, and the The results of lesion vascularity evaluated with CD31 negative control was a primary antibody replaced by buffer. Sec- ranged from a low of 7 vessels per high-powered field in tions of myometrium (blood vessels) were used as the positive the indeterminate leprosy lesions to a high of 68.5 in control for CD31. Diaminobenzidine was the chromogen used, lepromatous leprosy lesions. The mean±SEM CD31 and Dako automation hematoxylin was used as a counterstain microvessel density was 25.34±3.70 vessels per high- powered field in the borderline tuberculoid lesions, 36.25±5.20 in the midborderline lesions, and 60 ∗ 44.0±9.80 in the lepromatous lesions. These numbers 50 were significantly higher (P=.02, .004, and .03, respec- ∗ tively) when compared with a mean of 13.37±3.68 ves- 40 sels per high-powered field in indeterminate leprosy ∗ 30 lesions. The mean CD31 microvessel densities in the different leprosy types are shown in Figure 1.Aϫ20 20

Mean CD31 Count high-power view of indeterminate leprosy with a mean Figure 2 10 CD31 value of 7 is shown in A. For this patient, a total of 3 hot spots with 8, 6, and 7 vessels 0 Indeterminate Tuberculoid Borderline Borderline Borderline Lepromatous were counted, and the mean number of microvessels in Tuberculoid Lepromatous 2 hot spots was 7.5. Figure 2B shows a borderline lep- Leprosy Type romatous lesion, with a mean CD31 value of 39. For this slide, a total of 5 hot spots with values of 38, 31, Figure 1. Mean CD31 microvessel density for each leprosy type. Error bars 26, 40, and 23 were calculated, and the mean number indicate SEM. *PϽ.05 compared with indeterminate leprosy. of microvessels in 2 hot spots was 39.

A B

Figure 2. Immunohistochemical staining for CD31 in different stages of leprosy. A, High-power view (original magnification ϫ20) of an indeterminate leprosy lesion stained with CD31 polyclonal antibody and diaminobenzidine as the chromogen. A low microvessel count (7.5, which indicates a mean number of microvessels in 2 hot spots) is observed in the dermis. B, High-power view (original magnification ϫ20) of a borderline lepromatous lesion stained with monoclonal anti-CD31 antibody and diaminobenzidine as the chromogen. A high microvessel count (39, which indicates a mean number of microvessels in 2 hot spots) is observed in the dermis.

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 COMMENT of the manuscript: Bhandarkar, Kuruvila, MacKelfresh, Modlin, and Arbiser. Critical revision of the manuscript for important intellectual content: Cohen, Rea, Lee, Modlin, Leprosy or Hansen disease is a chronic infectious disease and Arbiser. Statisical analysis: Bhandarkar. Obtained fund- that primarily affects the peripheral nerves and the skin. ing: Arbiser. Administrative, technical, or material sup- Mycobacterium leprae has a unique predilection for Schwann port: Bhandarkar, Cohen, Kuruvila, Rea, Lee, Modlin, and cells20 and replicates inside the Schwann cells slowly Arbiser. throughout the years.21 The clinical response of the sus- Financial Disclosure: None reported. ceptible patient forms a spectrum according to the type of Funding/Support: Dr Arbiser was supported by National host immune response to M leprae and forms the basis of Institutes of Health grants RO1 AR47901 and RO1 AR02030 various classifications of leprosy. Ridley and Jopling7 clas- and Emory Skin Disease Research Core Center Grant P30 sified the clinical response into tuberculoid, borderline tu- AR42687, a Veterans Administration Hospital Merit Award, berculoid, borderline, borderline lepromatous, and lepro- also from the National Institutes of Health. matous leprosy. 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