The present study investigated the effects of a 12-week programme of lower-limb aerobic and resistance exercise on lower-limb cutaneous microvascular reactivity in adults with unilateral venous ulceration. The principal findings are as follows: (1) at baseline, the peak CVC responses to the endothelium-dependent vasodilator ACh and the endothelium-independent vasodilator SNP were depressed in ulcerated legs compared with non-ulcerated legs; (2) 12 weeks of exercise training improved the peak CVC responses to both ACh and SNP in the ulcerated leg, and to ACh (but not SNP) in the non-ulcerated leg; (3) the between-limb differences in cutaneous microvascular reactivity that were observed at baseline were no longer apparent in the exercise group at 3 months, and; (4) peak CVC responses to both ACh and SNP were greater in healed versus non-healed legs at 3 months.
The microvascular reactivity data were obtained using the reproducible and validated method of iontophoresis to deliver ACh and SNP to the skin microvasculature in a controlled manner without trauma, and laser Doppler fluxmetry to assess microvascular erythrocyte flux (Morris and Shore 1996; Berghoff et al. 2002; Tew et al. 2010). Although the galvanic effect of current and voltage application with iontophoresis can cause non-specific vasodilation (Berghoff et al. 2002), the use of 1% drug concentrations and currents ≤ 0.3 mA should have minimised such effects (Droog et al. 2004). In addition, as the protocol was consistent between groups before and after the intervention period, it is unlikely that any observed between-limb differences or changes in microvascular responses are due to non-specific, protocol-related vasodilatory effects.
The CVC response to ACh was used to assess microvascular endothelial function in vivo, whereas the CVC response to SNP was used to assess microvascular smooth muscle function (Morris and Shore 1996). ACh interacts with the M3 muscarinic receptor on the endothelial cell surface, which initiates a sequence of intracellular events—G protein activation, phospholipase A and/or C activation and stimulation of endothelial nitric oxide (NO) synthase activity—leading to NO synthesis, although prostacyclin and hyperpolarising factor release may also be induced (Berghoff et al. 2002; Holowatz et al. 2005; Kellogg et al. 2005; Fujii et al. 2013; Brunt et al. 2015). The NO diffuses across the endothelial cell membrane and basement membrane, binds to guanylate cyclase within the vascular smooth muscle cell, leading to an increase in intracellular cyclic guanosine monophosphate and, ultimately, smooth muscle relaxation and vasodilation. By contrast, SNP decomposes to release NO in vivo, which interacts with the vascular smooth muscle guanylate cyclase to produce vasodilation in an endothelium-independent manner (Turner et al. 2008).
The depressed vascular responses to ACh and SNP in ulcerated versus non-ulcerated legs (at baseline), therefore, indicate that aspects of cutaneous microvascular endothelial function and smooth muscle responsiveness to NO are disturbed in limbs with venous ulceration. Although the ACh and SNP data are consistent with that of previous work in earlier stage venous disease (Klonizakis et al. 2003), they contradict the findings of Ardron et al. (1991) who reported no significant differences in blood flux responses to ACh and SNP on the dorsum of the foot between 15 people with venous leg ulcers and 15 age and sex-matched healthy controls. The reasons underpinning these discrepant findings are unclear, but might be due to differences in study design; specifically in relation to the measurement site (gaiter region versus foot dorsum) and comparator (within-participant comparison of ulcerated and non-ulcerated legs versus between-participant comparison of patient and control feet). Other work by Jünger et al. (2000) has shown that cutaneous vascular reserve, as determined by the increase skin blood flow after a 3-min arterial occlusion (i.e., post-occlusive reactive hyperaemia), is exhausted at the site of a venous ulcer (healthy control group: + 721 ± 490%; in the ulcer: + 20 ± 41%, P < 0.01). Together, the available data suggest that lower-limb cutaneous microvascular reactivity is markedly impaired in people with advanced venous disease, at least in the gaiter region where most venous ulcers occur.
The mechanisms of cutaneous microvascular dysfunction in limbs with venous disease/ulceration remain unclear, although several possibilities exist. In venous disease, venous stasis in the microcirculation results in low shear stress on the endothelium, which leads to endothelial glycocalyx disruption and activation of endothelial cells and leucocytes by a variety of adhesion molecules and leucocyte chemoattractants (Raffetto 2016). This causes a decrease in endothelial NO synthase mRNA and protein expression that limits the bioavailability of NO (Qiu et al. 2013) and, potentially because of this, the vascular responsiveness to ACh. Alternatively, the impaired microvascular reactivity might be due to the localised persistent inflammatory state that is characteristic of venous ulcers (Raffetto 2016). Indeed, studies in rheumatoid arthritis have demonstrated that C-reactive protein (a blood marker of inflammation) correlates negatively with both endothelial-dependent and endothelial-independent microvascular function (Galarraga et al. 2008; Foster et al. 2010). Venous hypertension also causes microvascular structural abnormalities including a decrease in the number of capillaries and glomerulus-like changes in capillary morphology (Jünger et al. 2000), which might impact upon microvascular responsiveness to ACh and SNP. It should be noted that blood flow responses in the microcirculation assessed by laser Doppler are to a certain degree dependent on microvascular structure (Gardner-Medwin et al. 1997), so we cannot safely say that the between-limb differences in ACh and SNP responses are not influenced by vascular structural alterations. However, baseline (i.e., pre-stimulation) flux, which is a crude indicator of microvascular structure (Gardner-Medwin et al. 1997), was similar between ulcerated and non-ulcerated legs (Figs. 1, 2). It could also be argued that skin thickening due to lipodermatosclerosis seen in limbs with venous hypertension inhibited the iontophoretic delivery of ACh and SNP into the cutaneous circulation, thus reducing the amount of chemical available to initiate vasodilation. To minimise this possibility, we performed measurements away from the ulcer site, avoiding thickened skin or areas of oedema.
The principal novel finding of this study was that a 12-week supervised exercise programme improved measures of cutaneous microvascular reactivity in people being treated with compression therapy for venous ulceration. The data are broadly consistent with the results of a recent meta-analysis of seven controlled trials (n = 245), which showed that aerobic exercise training had a moderate and statistically significant beneficial effect (standardised mean difference = 0.43, 95% CI 0.08–0.78, P = 0.013) on cutaneous microvascular reactivity in a mixed population of adults who were primarily inactive at baseline (Lanting et al. 2017). Of the three trials that had used iontophoresis or microdialysis with ACh to assess cutaneous microvascular endothelial function (Klonizakis et al. 2009; Middlebrooke et al. 2006; Black et al. 2008), two reported a beneficial effect of training (Klonizakis et al. 2009; Black et al. 2008). Interestingly, our findings also demonstrated that a training effect was apparent for both ulcerated and non-ulcerated limbs, which may indicate both a therapeutic and preventative role of exercise training. The greater improvements observed in ulcerated legs might be due to a relatively depressed endothelial function at baseline (i.e., greater room for improvement).
The endothelial-dependent pathways and signalling events responsible for the improvement in ACh-induced vasodilation are largely unknown; however, an increase in NO bioavailability is a particularly plausible contributory factor given that aerobic exercise training (thrice weekly, 30 min at 30% heart rate reserve) has been shown to increase the NO-component of ACh-induced vasodilation in previously sedentary, healthy older adults (Black et al. 2008). Previous work indicates that improvements in NO-mediated vasodilator function might occur via the episodic increases in endothelial shear stress that occur with repeated bouts of exercise (Green et al. 2010). Shear stress upregulates endothelial NO synthase production in conduit arteries (Hambrecht et al. 2003), and Green et al. (2010) demonstrated that restricting increases in shear stress during an 8-week programme of repeated skin heating (bilateral forearm immersion in 42 °C water for 30 min, thrice weekly; an experimental model used to simulate exercise-induced thermoregulatory vasodilation) prevented improvements in conduit artery and cutaneous microvascular function from occurring. It is likely that the increases in cutaneous blood flow that occur acutely during exercise are due to a combination of thermoregulatory vasodilation and the direct action of the calf muscle pump (Eze et al. 1996; Simmons et al. 2011). Increasing core temperature and activating the calf muscle pump were therefore, important considerations during the design of the exercise programme. The focus on lower-limb exercise training was also informed by our previous studies showing that lower-limb endurance training (Klonizakis et al. 2009), but not upper-limb endurance training (Klonizakis et al. 2010), improved lower-limb cutaneous microvascular reactivity in post-surgical varicose vein patients.
That exercise training also improved the vascular responsiveness to SNP in ulcerated legs was a somewhat surprising finding given that several studies have shown no significant effect (Klonizakis et al. 2009; Tew et al. 2010; Wang 2005), including our aforementioned lower-limb exercise training study in post-surgical varicose vein patients (Klonizakis et al. 2009). However, Hodges et al. (2010) demonstrated that SNP-responsiveness was increased after 36 weeks of exercise training in post-menopausal women. Therefore, it may be that exercise training improves cutaneous vascular smooth muscle responsiveness to NO after longer periods of training (e.g., > 36 weeks), or that adaptations only occur in individuals with sub-optimal function at baseline. The latter might be more relevant to the present study given the shorter (12 week) programme duration, the fact that SNP responses were lower in ulcerated versus non-ulcerated legs at baseline, and that exercise training did not cause a significant improvement in SNP-responsiveness in non-ulcerated legs.
The role of impaired cutaneous microvascular reactivity in the pathogenesis of venous ulceration and the clinical significance of the exercise-induced improvements in cutaneous microvascular reactivity requires further research. For logistical reasons we were unable to perform additional interim microvascular assessments to explore if changes in cutaneous microvascular reactivity preceded ulcer healing. Although several factors are thought to influence susceptibility to ulceration and ulcer healing (Raffetto 2016), the functional integrity of the microcirculation to maintain blood flow, tissue oxygenation, and nutrient delivery is likely to be particularly important (Iabichella et al. 2006). This is supported by the work of Jünger et al. (2000), which indicated that various microvascular adaptations precede and determine ulcer healing. The generally large effect sizes and the fact that microvascular reactivity was superior in healed versus non-healed limbs at the 3-month assessment also support the notion that the observed changes may at least play some role in facilitating ulcer healing.
Strengths of this study include the randomised controlled study design, the low rates of attrition and missing data, the objective assessment of cutaneous microvascular reactivity, and the close adherence to the exercise and usual care interventions. Limitations include the relatively small sample size (which precluded detailed investigation of the relationship between exercise-induced improvements in cutaneous microvascular reactivity and ulcer healing), the short-term follow-up (which precluded investigation of cutaneous microvascular adaptations occurring after the exercise programme), and a lack of data exploring the mechanisms underpinning the observed differences and changes in microvascular responsiveness to ACh and SNP. Regarding the latter, further research is warranted to determine the extent to which vascular structural changes in venous disease influence the non-invasive estimates of microvascular function and their clinical significance. An additional limitation was that a lower-extremity venous duplex evaluation was not conducted at baseline to characterise each participant’s venous insufficiency. Nevertheless, the results are representative of what might be expected in a mixed outpatient population.
In summary, we observed an impairment of cutaneous microvascular reactivity in the gaiter area of legs with venous ulcers that was abolished following a 12-week programme of aerobic and resistance exercise. Although the precise mechanisms of these vascular adaptations are unclear, it is possible that exercise training-induced improvements in lower-limb cutaneous microvascular reactivity may facilitate the healing of venous leg ulcers. An appropriately powered trial is needed to clarify the effects of adjunctive exercise training on the healing rates of venous ulcers and the role of different microvascular adaptations in the healing process.