Vascular inflammation : implications for microvascular reconstructive surgery after irradiation

Better treatment has led to a rapidly increasing population of cancer survivors. A growing body of clinical evidence has shown that radiotherapy is associated with adverse effects on the cardiovascular system, such as myocardial infarction and stroke, depending on the previous treatment site. However, there is a paucity of experimental evidence linked to these clinical findings since the pathology, not evident until years after exposure, precludes adequate investigation through cell- and animal-experiments.

We present a differential global gene expression strategy, by comparing irradiated with nonirradiated conduit arteries and veins, harvested simultaneously from the same patient during microvascular free tissue transfers for cancer reconstruction. We could thereby benefit from the true advantages of microarray technology, bypassing the common problem of inter-individual variability and furthermore exclude the influence of other risk factors and study the effect of irradiation only. Surgery at different time-points after radiotherapy did furthermore give us the opportunity to study temporal aspects, a key-factor for the understanding of delayed vascular disease. Temporal aspects of vascular alterations caused by irradiation are furthermore of importance for the timing of surgery in relation to radiotherapy, since there has been a debate about treatment order and timing between the two.

In paper I, we could demonstrate that preoperative, compared to postoperative, radiotherapy was associated with microvascular occlusion after autologous free tissue transfers for head and neck reconstructions, and furthermore increased with the time elapsed from last radiotherapy session to surgery. In paper II, we utilized Affymetrix® microarray technology to unravel gene expression patterns in irradiated, compared to non-irradiated, arteries. Based on Gene Ontology Tree Machine®- analysis, target genes were selected and further confirmed with RT-PCR and immunohistochemistry. A major part of differentially expressed genes related to increased NF-kappaB activation, confined to cells within the arterial wall. The observed NF-kappaB activation, together with invading macrophages and T-cells, was evident even years after radiation exposure.

Since microvascular occlusions after free tissue transfers are more likely to occur on the venous side, further analyses were performed in veins in paper III, utilizing a Taqman® tissue low density array, including 45 selected target-genes involved in inflammation and coagulation. An acute NF-kappaB activation was detected in irradiated veins, confined to the endothelium, whereas in contrast to arteries, no sustained NF-kappaB activity was observed more than 15 weeks from last radiotherapy session. Neither was any detectable invasion of inflammatory cells observed. Immunohistochemistry indicated decreased staining of endothelial nitric oxide synthase (eNOS) in irradiated veins, compared to controls, in further support for an endothelial dysfunction caused by irradiation. A sustained activation was detected for plasminogen activator-1 (PAI-1) in irradiated veins. In study IV, we detected a decreased eNOS activity in endothelial cells after incubation with the free fatty acids (FFAs) palmitic and oleic, but not linolenic, acid, whereas a triglyceride-rich fat emulsion increased the eNOS activity. This is interesting since FFAs are markedly elevated during surgery.

With support from clinical and experimental data, we clearly advocate postoperative radiotherapy for microvascular reconstructive surgery, whenever possible for oncological reasons. Vascular inflammation may, together with increased PAI-1 gene expression observed in radiated veins, explain the increased risk for vascular complications when radiotherapy is administered prior to microvascular surgery. Moreover, the finding of a sustained NF-kappaB activation, together with presence of macrophages and T-cells, in irradiated arteries supports radiotherapy as an independent risk factor for cardiovascular disease and contributes to the search for therapeutic adjuncts to cope with the adverse effects of radiotherapy.

List of scientific papers

I. Halle M, Bodin I, Tornvall P, Wickman M, Farnebo F, Arnander C (2009). "Timing of radiotherapy in head and neck free flap reconstruction--a study of postoperative complications." J Plast Reconstr Aesthet Surg 62(7): 889-95.
https://pubmed.ncbi.nlm.nih.gov/18440289

II. Halle M, Gabrielsen A, Paulsson-Berne G, Gahm C, Agardh H, Farnebo F, Tornvall P (2010). "Sustained inflammation due to NF-kappaB activation in irradiated human arteries." J Am Coll Cardiol. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/20298930

III. Halle M, Ekström M, Farnebo F, Tornvall P (2010). "Endothelial activation with prothrombotic response in radiated microvascular recipient veins." J Plast Reconstr Aesthet Surg. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/20079702

IV. Halle M, Eriksson P, Tornvall P (2005). "Effects of free fatty acids and a triglyceride-rich fat emulsion on endothelial nitric oxide synthase." Eur J Clin Invest.
https://pubmed.ncbi.nlm.nih.gov/15667588