Collateral development in limb ischemia : aspects of endogenous and stimulated arteriogenesis

Peripheral arterial disease (PAD) is common among elderly and affects about 20% of the population above the age of 60 years. PAD is a strong marker for future cardiovascular events and increased mortality due to widespread atherosclerotic lesions in the coronary and cerebral arteries. Intermittent claudication (IC) with lower extremity pain on walking has a prevalence of 3-6 % in men at the age of 60-70 years according to large population studies.

Among these patients the majority remain stable without progression of symptoms. The symptoms progress in about 25% of the patients and distal ulceration or gangrene and ischemic rest pain, i.e critical limb ischemia (CLI) develops. Despite liberal indications for surgical or endovascular revascularizations, major amputation is required in as much as one third of the patients with CLI and for these patients there is a great need for alternative treatment strategies.

Collateral development, bridging stenoses or occlusions, is one of the reparative processes that reduce the consequences of coronary artery disease and PAD. The growth of these collaterals from pre-existing arteriole is called arteriogenesis. Angiogenesis, the sprouting of new capillaries, may also be of importance in the pathophysiology of PAD. There is little data establishing any impact of angiogenesis on PAD but it is likely that an angiogenic process occurs in ischemic tissues as a response to acute ischemia. Accordingly, hemodynamic forces, cytokines and inflammatory processes appear to be of importance in arteriogenesis, but the exact role of these processes is not firmly established. The arteriogenic mechanisms studied in animal models of limb ischemia and in vitro models aiming to stimulate arteriogenesis by supplying vascular growth factors have shown promising results.

The aim of this thesis was to assess the role of vascular growth factors in the arteriogenic process in PAD patients, and furthermore to evaluate gene therapy of FGF-4 in an animal model and humans as a method for therapeutic arteriogenesis.

First we explored the expression of growth factors in skin and muscle samples from 25 patients with CLI. There were increased levels of FGF-2 but not VEGF in distal muscle compared to proximal. Furthermore the expression of growth factors and inflammatory cytokines was investigated in 20 patients with IC after walking provocation. The proinflammatory cytokine IL-6 was significantly elevated one hour after exercise. VEGF mRNA also increased significantly in ischemic muscle samples after exercise.

Taken together these results indicate that VEGF expression rises locally in response to maximal exercise to the absolute limit of pain in patients with IC but not in patients with CLI at rest. FGF-2 seems to be upregulated in distal muscle in CLI patients which could be due to local inflammatory processes. Gene therapy with adenovirus mediated FGF-4 was assessed in a rat model of severe limb ischemia and in a phase I/II multicenter clinical trial in CLI patients. No signs of stimulated arteriogenesis were detected in the animal model 4 weeks after gene transfer. Gene therapy in 13 patients seemed safe, but no sign of stimulated arteriogenesis was able to be detected in this small patient cohort.

List of scientific papers

I. Palmer-Kazen U, Wariaro D, Luo F, Wahlberg E (2004). Vascular endothelial cell growth factor and fibroblast growth factor 2 expression in patients with critical limb ischemia. J Vasc Surg. 39(3): 621-8.
https://pubmed.ncbi.nlm.nih.gov/14981457

II. Palmer Kazen U, Religa P, Wahlberg E (2006). Expression of inflammatory cytokines and vascular growth factors following maximal treadmill exercise with intermittent claudication. [Manuscript]

III. Palmer Kazen U, Wariaro D, Rubyani GM, Groetzbach G, Wahlberg E (2006). AdFGF-4 gene therapy in a rat model of chronic limb ischemia. [Manuscript]

IV. Matyas L, Schulte KL, Dormandy JA, Norgren L, Sowade O, Grotzbach G, Palmer-Kazen U, Rubanyi GM, Wahlberg E (2005). Arteriogenic gene therapy in patients with unreconstructable critical limb ischemia: a randomized, placebo-controlled clinical trial of adenovirus 5-delivered fibroblast growth factor-4. Hum Gene Ther. 16(10): 1202-11.
https://pubmed.ncbi.nlm.nih.gov/16218781