Helicobacter pylori Prevents Proliferative Stage of Angiogenesis In Vitro Role of Cytokines LESLEY JENKINSON, BSc Hons,* KARNA DEV BARDHAN, MD,† JOHN ATHERTON, MD,‡ and NEENA KALIA, PhD* Inhibition of angiogenesis may explain the delayed ulcer healing following Helicobacter pylori infection. We have previosuly demonstrated that H. pylori can inhibit endothelial cell proliferation. Some cytokines possess antiangiogenic properties. This study assessed a role for IL-6, IL-8, and TNF- in H. pylori-induced endothelial cytostasis. First, 30 l of H. pylori was coincubated with microvascular endothelial cells in the presence or absence of monoclonal antibodies to IL-6, IL-8, and TNF- for 24, 48, 72, or 96 hr. Dual labeling with propidium iodide and Hoescht 33342 distinguished between necrosis, and apoptosis and allowed viable cell numbers to be determined. H. pylori decreased cell viability after 72 and 96 hr ( P  0.02). Neither necrosis nor apoptosis was observed. Monoclonal antibodies to IL-6 and IL-8 did not reverse cytostasis. However, significant MVEC proliferation was observed in the presence of the TNF- monoclonal antibody. In conclusion, H. pylori induces cytokine up-regulation as part of its pathophysiological mechanism, which could prove detrimental to ulcer healing through an inhibitory effect on angiogenesis. KEY WORDS: Helicobacter pylori; angiogenesis; endothelial cells; cytostasis; cytokines; proliferation. There is compelling evidence for the pivotal role of Helicobacter pylori in the pathogenesis of gastritis and peptic ulcer disease (1). However, delayed healing of established ulcers has also been frequently observed with H. pylori infection, with eradication of the bac- terium leading to improved healing rates (2– 4). Ex- perimentally, acid-induced ulcers in mice demon- strate a significantly prolonged ulcer healing time when exposed to extracts of H. pylori (5– 8). These studies collectively suggest that the healing of an ulcer is deleteriously influenced by continuous infection. Angiogenesis, or new blood vessel formation, is a fundamental biological process that occurs during wound and ulcer healing (9). It serves to reconstruct damaged vasculature and also increase blood flow to injured areas. Without restoration of blood flow, ox- ygen and nutrients cannot be delivered to the ulcer site, resulting in delayed healing (9). The process of angiogenesis occurs in a number of sequential stages (10). First, vessel endothelial cells respond to specific signals and undergo digestion of their basal lamina by proteolytic enzymes. Secondly, chemotactic factors, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), induce endo- thelial cell migration towards the signal origin. Endo- thelial cells then proliferate due to stimulation by mitogenic factors including VEGF and bFGF. Fi- nally, they form a tube or capillary-like structures that Manuscript received November 6, 2001; accepted February 15, 2002. From the *Section of Surgical and Anaesthetic Sciences, Divi- sion of Clinical Sciences (S), Royal Hallamshire Hospital, Sheffield, †District General Hospital, Rotherham; and ‡University Hospital, Queens Medical Centre, Nottingham, UK. This study was supported by Bardhan Research and Education Trust, Registered charity number: 328452. Address for reprint requests: Correspondence to: Dr. Neena Kalia, Academic Surgical Unit, Section of Surgical and Anaesthetic Sciences, K-Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, UK. Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002), pp. 1857–1862 (© 2002) 1857 Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002) 0163-2116/02/0800-1857/0 © 2002 Plenum Publishing Corporation