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Background: Hypertension is one of the most common diseases in the United Kingdom and it remains an important risk factor for cardiovascular morbidity and mortality. Dietary sodium is an important trigger for hypertension and humans show a heterogeneous blood pressure (BP) response to salt intake. The mechanisms for this have not been fully explained, with renal sodium handling thought to play a central role. Animal studies have shown that dietary salt loading results in Na+ accumulation and lymphangiogenesis in skin mediated by vascular endothelial growth factor-C (VEGF-C), both attenuating the rise in BP. This represents an additional system for maintaining BP and volume homeostasis in response to salt load. The focus of this thesis is to determine whether these dermal mechanisms exist in humans. Methods: The technique of measuring skin Na+ and K+ using inductively coupled plasma optical emission spectrometry was developed in a pilot study of healthy adults. In a further study in healthy adults, the effects of dietary salt modulation on skin Na+, the effect of sex and the relationship between skin Na+ and haemodynamic parameters and plasma VEGF-C were studied. Skin Na+ concentrations were expressed as the ratio Na+:K+ to correct for variability in sample hydration. The effect of dietary salt intake on skin gene expression of factors that potentially influence BP such as VEGF-C and the hypoxia inducible factor (HIF) transcription system was assessed, exploring possible mechanisms linking skin Na+ to haemodynamic variables. Results: Skin Na+:K+ increased with dietary salt loading and this effect appeared to be greater in men while only women showed a rise in ambulatory mean BP. Skin Na+:K+ correlated with blood pressure, stroke volume and peripheral vascular resistance in men, but not in women. No change was noted in plasma vascular endothelial growth factor-C. Conclusions: These findings suggest that the skin may buffer dietary Na+, reducing the hemodynamic consequences of increased salt and this may be influenced by sex. Skin Na+ may influence blood pressure, stroke volume and PVR.

Chronic wound healing has a continuously increasing prevalence in elderly and diabetic patients. The long-lasting non-healing properties of chronic wounds present a strain to the quality of life of patients and to costs in health care. Almost three decades ago it was discovered that lymphatic function is impaired, and vessels are largely absent in chronic ulcers. Thus, there is a large knowledge gap in understanding the pathogenesis of chronic wounds, which represents a major therapeutic challenge. In the main project of this thesis, we investigated the biological role of lymphatic vessels in wound repair and hypothesized that targeted lymphatic activation might improve the healing and quality of diabetic wounds. To this end, full-thickness excisional wound healing studies were conducted in wildtype mice, transgenic mice with an increased number of cutaneous lymphatic vessels (K14-VEGF-C), and transgenic mice that lack lymphatic vessels in the skin (K14-sVEGFR3). Furthermore, diabetic mice (db/db) were treated i.v. either with a recombinant F8-VEGF-C fusion protein, targeting the vascular endothelial growth factor (VEGF)-C protein to wounds via binding to the EDA fibronectin splice variant, which we found to be upregulated in diabetic wounds, or with a control protein (F8-SIP). Lymphangiogenesis was detected at the edge of the wound and within the granulation tissue between 7- and 10-days post-wounding, was increased in K14-VEGF-C transgenic mice and was completely absent in K14-sVEGFR3 mice. These data agreed with the up- and downregulation of lymphatic-related genes as evaluated by RNAseq. The lack of cutaneous lymphatic vessels was associated with delayed wound closure in K14-sVEGFR3 mice, whereas inhibition of VEGFR3 with a blocking antibody completely prevented lymphangiogenesis. The increase in lymphatic density was accompanied by a decrease in myofibroblast density in K14-VEGF-C mice. In db/db mice, targeted delivery of VEGF-C induced lymphangiogenesis and improved wound healing. Increased lymphatic density was associated with a reduced level of inflammatory cells and an increase in myofibroblast and collagen I density in diabetic wounds. In a second project, our objective was to study the advantages of an optoacoustic imaging technique to assess blood microvascular alterations in skin wounds. Longitudinal monitoring of angiogenesis in vivo remains challenging, and injections of tracers remain common. To this end, a large-scale optoacoustic microscopy was used to non-invasively image wound repair in K14-VEGF-A transgenic mice with an increased blood microvascular density in the skin. We detected increased vascularization in healing wounds and assessed vessel diameter, tortuosity, hemoglobin content and fill fraction and obtained comparable results to classical histology. Together, our findings demonstrate that lymphatic vessels play a vital role in wound repair and that activation of lymphatic vessels with the F8-VEGF-C fusion protein improves the quality of diabetic wounds, indicating a potential new strategy for treating diabetic wounds in the future. Finally, the new large-scale optoacoustic microscopy approach demonstrates the possibility to non-invasively image angiogenesis in wound repair.