Russell H. Mellor

Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis

Lymphatic vessels are essential for the removal of interstitial fluid and prevention of tissue edema. Lymphatic capillaries lack associated mural cells, and collecting lymphatic vessels have valves, which prevent lymph backflow. In lymphedema-distichiasis (LD), lymphatic vessel function fails because of mutations affecting the forkhead transcription factor FOXC2. We report that Foxc2−/− mice show abnormal lymphatic vascular patterning, increased pericyte investment of lymphatic vessels, agenesis of valves and lymphatic dysfunction. In addition, an abnormally large proportion of skin lymphatic vessels was covered with smooth muscle cells in individuals with LD and in mice heterozygous for Foxc2 and for the gene encoding lymphatic endothelial receptor, Vegfr3 (also known as Flt4). Our data show that Foxc2 is essential for the morphogenesis of lymphatic valves and the establishment of a pericyte-free lymphatic capillary network and that it cooperates with Vegfr3 in the latter process. Our results indicate that an abnormal interaction between the lymphatic endothelial cells and pericytes, as well as valve defects, underlie the pathogenesis of LD.

Mutations in FOXC2 Are Strongly Associated With Primary Valve Failure in Veins of the Lower Limb

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P<0.0001, Fisher exact test). Deep vein reflux was recorded in 14 of 18 participants. Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.

Differences in lymph drainage between swollen and non-swollen regions in arms with breast-cancer-related lymphoedema

Recent research indicates that the pathophysiology of breast-cancer-related lymphoedema (BCRL) is more complex than simple axillary lymphatic obstruction as a result of the cancer treatment. Uneven distribution of swelling (involvement of the mid-arm region is common, but the hand is often spared) is puzzling. Our aim was to test the hypothesis that local differences in lymphatic drainage contribute to the regionality of the oedema. Using lymphoscintigraphy, we measured the removal rate constant, k (representing local lymph flow per unit distribution volume, VD), for 99mTc-labelled human immunoglobulin G in the oedematous proximal forearm, and in the hand (finger web) in women in whom the hand was unaffected. Tracer was injected subcutaneously, and the depot plus the rest of the arm was monitored with a gamma-radiation camera for up to 6 h. VD was assessed from image width. Contralateral arms served as controls. k was 25% lower in oedematous forearm tissue than in the control arm (BCRL, -0.070+/-0.026% x min(-1); control, -0.093+/-0.028% x min(-1); mean+/-S.D.; P=0.012) and VD was greater. In the non-oedematous hand of the BCRL arm, k was 18% higher than in the control hand (BCRL, -0.110+/-0.027% x min(-1); control, -0.095+/-0.028% x min(-1); P=0.057) and 59% higher than forearm k on the BCRL side (P=0.0014). VD did not differ between the hands. Images of the BCRL arm following hand injection showed diffuse activity in the superficial tissues, sometimes extending almost to the shoulder. A possible interpretation is that the hand is spared in some patients because local lymph flow is increased and diverted along collateral dermal routes. The results support the hypothesis that regional differences in surviving lymphatic function contribute to the distribution of swelling.

Dual-frequency ultrasound examination of skin and subcutis thickness in breast cancer-related lymphedema.

Abstract:  Breast cancer‐related lymphedema (BCRL) is a chronic swelling of the arm that sometimes follows breast cancer treatment. Clinically, both skin and subcutis are swollen. Edema is considered to be predominantly subcutaneous and of an even distribution. The purpose of this study was to quantify the degree and uniformity of skin and subcutis swelling around the forearms of women with BCRL. Ten women with BCRL were recruited. Both forearms were examined using 20 MHz ultrasound to visualize the skin and 7 MHz ultrasound to visualize the subcutis. Skin thickness was between the bottom of the entry‐echo and the skin–subcutis boundary. Subcutis thickness was measured between the skin–subcutis boundary and the subcutis–muscle boundary. Both average skin thickness (1.97 ± 1.00 mm) and average subcutis thickness (10.32 ± 5.63 mm) were greater in the ipsilateral arm than in the contralateral arm (skin 1.12 ± 0.14 mm, subcutis 5.58 ± 2.04 mm, p < 0.01, t‐test). The degree of increase in skin thickness did not vary around the arm (p > 0.05, ANOVA), while the degree of increase in subcutis thickness did vary (p < 0.05). Skin thickness correlated negatively with subcutis thickness in the contralateral arm, but correlated positively in the ipsilateral arm. The skin and subcutis are thickened in the ipsilateral arm of patients with BCRL. Skin thickness is increased uniformly around the arm and correlates strongly with the degree of swelling, while subcutis swelling varies. The measurement of skin thickness using ultrasound may form a useful clinical tool in the diagnosis of lymphedema and also aid further investigation of therapeutic techniques. 

Enhanced cutaneous lymphatic network in the forearms of women with postmastectomy oedema.

Postmastectomy oedema (PMO) of the arm is a common aftermath of axillary lymphatic damage during treatment for breast cancer. The aim of the present study was to quantify the forearm dermal lymphatic capillaries in order to determine whether they exhibit adaptive responses to PMO. Both forearms were examined by fluorescence microlymphography in 16 patients with oedema following treatment for breast cancer (mean swelling 25 ± 4%) and 19 patients treated for breast cancer but without oedema. Delineated lymphatic networks were analysed stereologically. The main findings were: (1) lymphatic density at any specified distance from the injection site was greater in the swollen arm than the control arm (p < 0.01, t test); (2) taking into account the increased skin area, the total length of lymphatic capillaries in a 1-cm annulus of skin was 676 ± 56 cm (swollen), compared with 385 ± 30 cm (control) (p < 0.001, t test); (3) fluorescent marker was transported over a greater distance before draining deep in the swollen arm (2.74 ± 0.33 cm) than in the control arm (1.59 ± 0.24 cm) (p = 0.02); (4) there was no evidence of lymphatic dilatation in the swollen arm, and (5) in breast cancer patients without swelling, the arm on the side of radiotherapy/surgery (otherwise referred to as the unswollen arm) showed none of the above changes, indicating that the changes are linked to the oedema rather than being universal responses to breast cancer or its treatment. It is concluded that microlymphatic changes occur in the swollen arm, namely a local superficial rerouting of lymph drainage and either lymphangiogenesis and/or increased recruitment of dormant lymphatic vessels. Since blood capillary angiogenesis occurs in the swollen arms, and lymphangiogenesis occurs in experimental lymphoedema, there is a precedent for proposing lymphangiogenesis in PMO. An increased number of functional vessels would help to maintain the ratio of local tissue drainage capacity to filtration capacity.

Lymphatic Dysfunction, Not Aplasia, Underlies Milroy Disease

Microcirculation (2010) 17, 281–296. doi: 10.1111/j.1549‐8719.2010.00030.x

Lymphoedema-distichiasis and FOXC2: unreported mutations, de novo mutation estimate, families without coding mutations

Lymphoedema-distichiasis (LD) is a syndromic form of primary lymphoedema, where mutations in the gene for the developmental transcription factor FOXC2 have been shown to be causative. The disorder has been considered very rare, but our group has now ascertained 34 families and 11 sporadic cases in the UK. Two families with LD have no mutation in the coding region of FOXC2, although both are consistent with linkage to the FOXC2 locus. A deletion has been ruled out as a possible cause of LD in these families, leaving promoter mutations as the most likely cause. Sixteen previously unpublished mutations are reported, plus an estimate of the frequency of new mutations in this disorder.

In vivo quantification of the structural abnormalities in psoriatic microvessels before and after pulsed dye laser treatment

Background  Microvascular abnormalities (capillary elongation, widening and tortuosity) are a characteristic feature of psoriasis and form one of the pathological diagnostic criteria. These changes occur early in the progression of a psoriatic plaque, before there is clinical or histological evidence of epidermal hyperplasia. Treatment of psoriatic microvessels with a pulsed dye laser (PDL) has been associated with both clinical improvement and clearance of lesions.

Mutations in FOXC2 in Humans (Lymphoedema Distichiasis Syndrome) Cause Lymphatic Dysfunction on Dependency

Background: Human lymphoedema distichiasis syndrome (LDS) results from germline mutations in transcription factor FOXC2. In a mouse model, lack of lymphatic and venous valves is observed plus abnormal smooth muscle cell recruitment to initial lymphatics. We investigated the mechanism of lymphoedema in humans with FOXC2 mutations, specifically the effect of gravitational forces on dermal lymphatic function. Methods: We performed (1) quantitative fluorescence microlymphangiography (FML) on the skin of the forearm (non-swollen region) at heart level, and the foot (swollen region) below heart level (dependent) and then at heart level, and (2) immunohistochemical staining of microlymphatics in forearm and foot skin biopsies, using antibodies to podoplanin, LYVE-1 and smooth muscle actin. Results: FML revealed a marked reduction in fluid uptake by initial lymphatics in the LDS foot during dependency, yet normal uptake (similar to controls) in the same foot at heart level and in LDS forearms. In control subjects, dependency did not impair initial lymphatic filling. Immunohistochemical microlymphatic density in forearm and foot did not differ between LDS and controls. Conclusions:FOXC2 mutations cause a functional failure of dermal initial lymphatics during gravitational stress (dependency), but not hypoplasia. The results reveal a pathophysiological mechanism contributing to swelling in LDS.

Evidence for Dermal Angiogenesis in Breast Cancer Related Lymphedema Demonstrated Using Dual-Site Fluorescence Angiography

Objective: To determine whether the skin expansion associated with breast cancer related lymphedema (BCRL) reduces microvessel density or causes a compensatory angiogenesis.