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Figure 1.
Mean CD31 microvessel density for each leprosy type. Error bars indicate SEM. *P < .05 compared with indeterminate leprosy.

Mean CD31 microvessel density for each leprosy type. Error bars indicate SEM. *P < .05 compared with indeterminate leprosy.

Figure 2.
Immunohistochemical staining for CD31 in different stages of leprosy. A, High-power view (original magnification ×20) 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 ×20) 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.

Immunohistochemical staining for CD31 in different stages of leprosy. A, High-power view (original magnification ×20) 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 ×20) 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.

1.
Padma  TV With scores still infected, India declares leprosy “eliminated.” Nat Med 2006;12 (4) 372
PubMedArticle
2.
Deps  PDGuedes  BVBucker Filho  JAndreatta  MKMarcari  RSRodrigues  LC Characteristics of known leprosy contact in a high endemic area in Brazil. Lepr Rev 2006;77 (1) 34- 40
PubMed
3.
Williams  MC How can adherence with multi-drug therapy in leprosy be improved? Lepr Rev 2005;76 (2) 160- 161
PubMed
4.
Ustianowski  APLawn  SDLockwood  DN Interactions between HIV infection and leprosy: a paradox. Lancet Infect Dis 2006;6 (6) 350- 360
PubMedArticle
5.
Cross  H Interventions to address the stigma associated with leprosy: a perspective on the issues. Psychol Health Med 2006;11 (3) 367- 373
PubMedArticle
6.
Ridley  DSJopling  WH A classification of leprosy for research purposes. Lepr Rev 1962;33119- 128
PubMed
7.
Ridley  DSJopling  WH Classification of leprosy according to immunity—a five-group system. Int J Lepr Other Mycobact Dis 1966;34 (3) 255- 273
PubMed
8.
Voest  EEKenyon  BMO’Reilly  MSTruitt  GD’Amato  RJFolkman  J Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 1995;87 (8) 581- 586
PubMedArticle
9.
Kim  JUyemura  KVan Dyke  MK  et al.  A role for IL-12 receptor expression and signal transduction in host defense in leprosy. J Immunol 2001;167 (2) 779- 786
PubMedArticle
10.
Libraty  DHAiran  LEUyemura  K  et al.  Interferon-gamma differentially regulates interleukin-12 and interleukin-10 production in leprosy. J Clin Invest 1997;99 (2) 336- 341
PubMedArticle
11.
Ueno  TToi  MSaji  H  et al.  Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 2000;6 (8) 3282- 3289
PubMed
12.
Drexler  HGUphoff  CCGaidano  GCarbone  A Lymphoma cell lines: in vitro models for the study of HHV-8 primary effusion lymphomas (body cavity-based lymphomas). Leukemia 1998;12 (10) 1507- 1517
PubMedArticle
13.
Hatanaka  HAbe  YNaruke  M  et al.  Significant correlation between interleukin 10 expression and vascularization through angiopoietin/TIE2 networks in non-small cell lung cancer. Clin Cancer Res 2001;7 (5) 1287- 1292
PubMed
14.
Arbiser  JLJohnson  DCohen  CBrown  LF High-level expression of vascular endothelial growth factor and its receptors in an aphthous ulcer. J Cutan Med Surg 2003;7 (3) 225- 228
PubMedArticle
15.
Tamargo  RJBok  RABrem  H Angiogenesis inhibition by minocycline. Cancer Res 1991;51 (2) 672- 675
PubMed
16.
Arbiser  JLFlynn  EBarnhill  RL Analysis of vascularity of human neurofibromas. J Am Acad Dermatol 1998;38 (6, pt 1) 950- 954
PubMedArticle
17.
Arbiser  JLBrat  DHunter  S  et al.  Tuberous sclerosis-associated lesions of the kidney, brain and skin are angiogenic neoplasms. J Am Acad Dermatol 2002;46 (3) 376- 380
PubMedArticle
18.
Weidner  NFolkman  JPozza  F  et al.  Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84 (24) 1875- 1887
PubMedArticle
19.
Macaron  NCCohen  CChen  SCArbiser  JL Cutaneous lesions of secondary syphilis are highly angiogenic. J Am Acad Dermatol 2003;48 (6) 878- 881
PubMedArticle
20.
Rambukkana  AYamada  HZanazzi  G  et al.  Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium lepraeScience 1998;282 (5396) 2076- 2079
PubMedArticle
21.
Britton  WJLockwood  DN Leprosy [review]. Lancet 2004;363 (9416) 1209- 1219
PubMedArticle
22.
Findlay  GH Indeterminate leprosy: a new clinical variety. Br J Dermatol 1951;63 (3) 100- 104
PubMedArticle
23.
Yawalkar  SJ Leprosy: For Medical Practitioners and Paramedical Workers. 7th ed. Basel, Switzerland Novartis Foundation for Sustainable Development2002;
24.
Mira  MT Genetic host resistance and susceptibility to leprosy. Microbes Infect 2006;8 (4) 1124- 1131
PubMedArticle
25.
Antunes  SLMotta  Ede Almeida  SMGallo  MENery  JALenzi  HL Distinct patterns of microvasculature in the cutaneous lesions of leprosy. Int J Lepr Other Mycobact Dis 2000;68 (2) 143- 151
PubMed
Study
December 2007

Angiogenesis in Cutaneous Lesions of LeprosyImplications for Treatment

Author Affiliations

Author Affiliations: Departments of Dermatology (Drs Bhandarkar, MacKelfresh, and Arbiser) and Pathology (Dr Cohen), Emory University School of Medicine, Atlanta, Georgia; Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles (Drs Lee and Modlin); Department of Dermatology, Venereology, and Leprology, Kasturba Medical College, Mangalore, India (Drs Bhandarkar and Kuruvila); and Department of Dermatology, University of Southern California, Los Angeles (Dr Rea).

Arch Dermatol. 2007;143(12):1527-1529. doi:10.1001/archderm.143.12.1527
Abstract

Objective  To examine the potential role of angiogenesis in leprosy.

Design  Immunohistochemical analysis of leprosy lesions.

Setting  Department of Dermatology, Venereology, and Leprology, Kasturba Medical College; Division of Dermatology, University of California at Los Angeles; and Departments of Dermatology and Pathology, Emory University.

Patients  Thirty-two cutaneous lesions that represented the spectrum of leprosy were obtained from 32 patients.

Main Outcome Measure  CD31 microvessel counts.

Results  The mean CD31 microvessel count in borderline tuberculoid, midborderline, and lepromatous leprosy lesions was significantly higher than in indeterminate leprosy lesions.

Conclusions  Increased bacterial load is associated with increased angiogenesis. Angiogenesis inhibitors may be of benefit in the treatment of leprosy.

Leprosy is a chronic infection caused by Mycobacterium leprae and is endemic to tropical areas of the world, including the Indian subcontinent,1 Indochina, and South America.2 Leprosy causes much morbidity through formation of trophic ulcers, eye disabilities, neuropathy, and social isolation. Although leprosy is currently treatable, treatment of leprosy requires long courses of multiple antibiotics, which can decrease compliance.3 During treatment, patients can experience debilitating erythema nodosum leprosum and reversal reactions.4 Finally, the irreversible neural damage and its associated sensory defects lead to the deformities and disabilities that form a part of the social stigma associated with leprosy.5

Leprosy exists in a clinical spectrum of disease, ranging from solitary inflammatory lesions called tuberculoid to disseminated disease characterized by formation of highly bacilliferous granulomas, which is termed lepromatous.6,7 Leprosy, like other mycobacterial infections (tuberculosis), leishmaniasis, and syphilis, requires intact cellular immunity for clearance.7 Interleukin 12 (IL-12) has been demonstrated to play a pivotal role in the clearance of these pathogens, and the clinical spectrum of leprosy likely reflects the ability of the host to mount an IL-12 response to the pathogen.7 IL-12 was shown to be a potent angiogenesis inhibitor, as were some of the cytokines induced by IL-12, including interferon-inducible protein 10 and interferon gamma.813 Thalidomide, a drug used to treat leprosy, also has antiangiogenic properties.14 Minocycline, a drug that is a part of the rifampin, ofloxacin, and minocycline regimen given for single skin lesions such as paucibacillary leprosy, has antiangiogenic properties through inhibition of matrix metalloproteinases.15 Given the potential role of angiogenesis in leprosy and also since M leprae, the causative organism of leprosy, cannot be cultured, we wanted to study the vascularity of the entire spectrum of leprosy through histologic assessment. Our findings demonstrate an increase in angiogenesis toward the lepromatous spectrum lesions and raise the possibility that angiogenesis inhibitors may be useful in the treatment of leprosy.

METHODS

We studied 32 cutaneous lesions that represented the spectrum of leprosy from 32 patients for expression of CD31. Twenty-eight paraffin blocks were received from the Department of Dermatology, Venereology, and Leprology, Kasturba Medical College, and 4 sections were received from the Division of Dermatology, University of California at Los Angeles. The diagnosis of leprosy in the patients was supported by hematoxylin-eosin–stained histologic analyses of the biopsied lesions and the clinical history of the patients. We studied 4 sections of indeterminate leprosy, 5 of tuberculoid leprosy, 12 of borderline tuberculoid leprosy, 3 of midborderline leprosy, 3 of borderline lepromatous leprosy, and 5 of lepromatous leprosy.

IMMUNOHISTOCHEMICAL ANALYSIS

A total of 32 sections (5 mm) of formalin-fixed, paraffin-embedded tissue (5 μm) were immunostained with monoclonal antibodies against CD31 (1:80) (clone JC170A, 1/80; Dako Corp, Carpinteria, California) using a horseradish peroxidase–labeled polymer, a heat-induced antigen retrieval, and an autostainer (Dako).16,17 The Dako Envision system is a 2-step horseradish peroxidase–labeled polymer that is conjugated with secondary antibodies and is used in combination with the automated Dako Autostainer. Hematoxylin was used as the counterstain, and the negative control was a primary antibody replaced by buffer. Sections of myometrium (blood vessels) were used as the positive control for CD31. Diaminobenzidine was the chromogen used, and Dako automation hematoxylin was used as a counterstain for 15 minutes. The mean CD31 microvessel density was quantitated microscopically by 2 independent observers (C.C. and J.B.M.). The number of CD31-positive blood vessels in the whole biopsy specimen and in 2 hot spots at a power of ×20 was determined according to the method of Weidner et al.18 Hot spots were areas determined by the observers to represent the fields of greatest vascular density within a given section.19 The mean number of microvessels in 2 hot spots from each section was determined, and the total mean of each spectrum was compared with indeterminate leprosy.

STATISTICAL ANALYSIS

P <.05 was considered statistically significant. A t test was used for statistical analysis, which was performed with Excel (Microsoft Inc, Redmond, Washington).

RESULTS

The results of lesion vascularity evaluated with CD31 ranged from a low of 7 vessels per high-powered field in the indeterminate leprosy lesions to a high of 68.5 in lepromatous leprosy lesions. The mean ± SEM CD31 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 44.0 ± 9.80 in the lepromatous lesions. These numbers were significantly higher (P = .02, .004, and .03, respectively) when compared with a mean of 13.37 ± 3.68 vessels per high-powered field in indeterminate leprosy lesions. The mean CD31 microvessel densities in the different leprosy types are shown in Figure 1. A ×20 high-power view of indeterminate leprosy with a mean CD31 value of 7 is shown in Figure 2A. For this patient, a total of 3 hot spots with 8, 6, and 7 vessels were counted, and the mean number of microvessels in 2 hot spots was 7.5. Figure 2B shows a borderline lepromatous lesion, with a mean CD31 value of 39. For this slide, a total of 5 hot spots with values of 38, 31, 26, 40, and 23 were calculated, and the mean number of microvessels in 2 hot spots was 39.

COMMENT

Leprosy or Hansen disease is a chronic infectious disease that primarily affects the peripheral nerves and the skin. Mycobacterium leprae has a unique predilection for Schwann cells20 and replicates inside the Schwann cells slowly throughout the years.21 The clinical response of the susceptible patient forms a spectrum according to the type of host immune response to M leprae and forms the basis of various classifications of leprosy. Ridley and Jopling7 classified the clinical response into tuberculoid, borderline tuberculoid, borderline, borderline lepromatous, and lepromatous leprosy. Patients with tuberculoid leprosy with strong cell-mediated immunity are at one end of the spectrum, and patients with lepromatous leprosy and poor cell-mediated immunity are at the other end of the spectrum. Indeterminate leprosy was first described as a new clinical variant by Findlay22 and was included in the World Health Organization 1952 classification. Indeterminate leprosy is often the clinical beginning of the disease, and approximately 80% of susceptible patients will show spontaneous regression of lesions, and 20% will progress to a more definitive form of leprosy.23,24 The role of immunologic responses in all the different forms of leprosy has been long established, but data are lacking on the role of angiogenesis in the spectrum of leprosy. We wanted to determine the role of angiogenesis, if any, in the entire spectrum of leprosy, including indeterminate leprosy.

Mycobacterium leprae has been found in endothelial cells of blood vessels, and anti–factor VIII–related antigen antibody has been used to demonstrate a difference in the microvascular pattern between the 2 ends of the spectrum, with lepromatous leprosy demonstrating a tortuous mesh of microvessels among the M leprae–laden macrophages; however, the microvessels in the tuberculoid lesions were restricted to the periphery of the granulomas.25 We studied the microvessel counts in the entire Ridley-Jopling spectrum and found an apparent overall increase in microvessel count toward lepromatous lesions compared with indeterminate leprosy lesions. The microvessel count was significantly higher in borderline tuberculoid lesions, midborderline lesions, and lepromatous lesions when compared with indeterminate lesions. We observed that the increase in microvessel count follows the same pattern of increase as M leprae loads toward the lepromatous end of the spectrum. We propose that new treatments, such as angiogenesis inhibitors directed toward leprosy, could potentiate the current multidrug treatment for leprosy.

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Article Information

Correspondence: Jack L. Arbiser, MD, PhD, Department of Dermatology, Emory University School of Medicine, WMB 5309, 1639 Pierce Dr, Atlanta, GA 30322 (jarbise@emory.edu).

Financial Disclosure: None reported.

Accepted for Publication: April 19, 2007.

Author Contributions: Dr Arbiser had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Bhandarkar and Arbiser. Acquisition of data: Bhandarkar, Cohen, Kuruvila, Rea, Lee, and Modlin. Analysis and interpretation of data: Bhandarkar, Cohen, MacKelfresh, and Arbiser. Drafting of the manuscript: Bhandarkar, Kuruvila, MacKelfresh, Modlin, and Arbiser. Critical revision of the manuscript for important intellectual content: Cohen, Rea, Lee, Modlin, and Arbiser. Statisical analysis: Bhandarkar. Obtained funding: Arbiser. Administrative, technical, or material support: Bhandarkar, Cohen, Kuruvila, Rea, Lee, Modlin, and Arbiser.

Funding/Support: Dr Arbiser was supported by National Institutes of Health grants RO1 AR47901 and RO1 AR02030 and Emory Skin Disease Research Core Center Grant P30 AR42687, a Veterans Administration Hospital Merit Award, also from the National Institutes of Health.

References
1.
Padma  TV With scores still infected, India declares leprosy “eliminated.” Nat Med 2006;12 (4) 372
PubMedArticle
2.
Deps  PDGuedes  BVBucker Filho  JAndreatta  MKMarcari  RSRodrigues  LC Characteristics of known leprosy contact in a high endemic area in Brazil. Lepr Rev 2006;77 (1) 34- 40
PubMed
3.
Williams  MC How can adherence with multi-drug therapy in leprosy be improved? Lepr Rev 2005;76 (2) 160- 161
PubMed
4.
Ustianowski  APLawn  SDLockwood  DN Interactions between HIV infection and leprosy: a paradox. Lancet Infect Dis 2006;6 (6) 350- 360
PubMedArticle
5.
Cross  H Interventions to address the stigma associated with leprosy: a perspective on the issues. Psychol Health Med 2006;11 (3) 367- 373
PubMedArticle
6.
Ridley  DSJopling  WH A classification of leprosy for research purposes. Lepr Rev 1962;33119- 128
PubMed
7.
Ridley  DSJopling  WH Classification of leprosy according to immunity—a five-group system. Int J Lepr Other Mycobact Dis 1966;34 (3) 255- 273
PubMed
8.
Voest  EEKenyon  BMO’Reilly  MSTruitt  GD’Amato  RJFolkman  J Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 1995;87 (8) 581- 586
PubMedArticle
9.
Kim  JUyemura  KVan Dyke  MK  et al.  A role for IL-12 receptor expression and signal transduction in host defense in leprosy. J Immunol 2001;167 (2) 779- 786
PubMedArticle
10.
Libraty  DHAiran  LEUyemura  K  et al.  Interferon-gamma differentially regulates interleukin-12 and interleukin-10 production in leprosy. J Clin Invest 1997;99 (2) 336- 341
PubMedArticle
11.
Ueno  TToi  MSaji  H  et al.  Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 2000;6 (8) 3282- 3289
PubMed
12.
Drexler  HGUphoff  CCGaidano  GCarbone  A Lymphoma cell lines: in vitro models for the study of HHV-8 primary effusion lymphomas (body cavity-based lymphomas). Leukemia 1998;12 (10) 1507- 1517
PubMedArticle
13.
Hatanaka  HAbe  YNaruke  M  et al.  Significant correlation between interleukin 10 expression and vascularization through angiopoietin/TIE2 networks in non-small cell lung cancer. Clin Cancer Res 2001;7 (5) 1287- 1292
PubMed
14.
Arbiser  JLJohnson  DCohen  CBrown  LF High-level expression of vascular endothelial growth factor and its receptors in an aphthous ulcer. J Cutan Med Surg 2003;7 (3) 225- 228
PubMedArticle
15.
Tamargo  RJBok  RABrem  H Angiogenesis inhibition by minocycline. Cancer Res 1991;51 (2) 672- 675
PubMed
16.
Arbiser  JLFlynn  EBarnhill  RL Analysis of vascularity of human neurofibromas. J Am Acad Dermatol 1998;38 (6, pt 1) 950- 954
PubMedArticle
17.
Arbiser  JLBrat  DHunter  S  et al.  Tuberous sclerosis-associated lesions of the kidney, brain and skin are angiogenic neoplasms. J Am Acad Dermatol 2002;46 (3) 376- 380
PubMedArticle
18.
Weidner  NFolkman  JPozza  F  et al.  Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84 (24) 1875- 1887
PubMedArticle
19.
Macaron  NCCohen  CChen  SCArbiser  JL Cutaneous lesions of secondary syphilis are highly angiogenic. J Am Acad Dermatol 2003;48 (6) 878- 881
PubMedArticle
20.
Rambukkana  AYamada  HZanazzi  G  et al.  Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium lepraeScience 1998;282 (5396) 2076- 2079
PubMedArticle
21.
Britton  WJLockwood  DN Leprosy [review]. Lancet 2004;363 (9416) 1209- 1219
PubMedArticle
22.
Findlay  GH Indeterminate leprosy: a new clinical variety. Br J Dermatol 1951;63 (3) 100- 104
PubMedArticle
23.
Yawalkar  SJ Leprosy: For Medical Practitioners and Paramedical Workers. 7th ed. Basel, Switzerland Novartis Foundation for Sustainable Development2002;
24.
Mira  MT Genetic host resistance and susceptibility to leprosy. Microbes Infect 2006;8 (4) 1124- 1131
PubMedArticle
25.
Antunes  SLMotta  Ede Almeida  SMGallo  MENery  JALenzi  HL Distinct patterns of microvasculature in the cutaneous lesions of leprosy. Int J Lepr Other Mycobact Dis 2000;68 (2) 143- 151
PubMed
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