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.

Lymphatic dysfunction, not aplasia, underlies Milroy disease.

OBJECTIVE: Milroy disease is an inherited autosomal dominant lymphoedema caused by mutations in the gene for vascular endothelial growth factor receptor-3 (VEGFR-3, also known as FLT4). The phenotype has to date been ascribed to lymphatic aplasia. We further investigated the structural and functional defects underlying the phenotype in humans. METHODS: The skin of the swollen foot and the non-swollen forearm was examined by (i) fluorescence microlymphangiography, to quantify functional initial lymphatic density in vivo; and (ii) podoplanin and LYVE-1 immunohistochemistry of biopsies, to quantify structural lymphatic density. Leg vein function was assessed by colour Doppler duplex ultrasound. RESULTS: Milroy patients exhibited profound (86-91%) functional failure of the initial lymphatics in the foot; the forearm was unimpaired. Dermal lymphatics were present in biopsies but density was reduced by 51-61% (foot) and 26-33% (forearm). Saphenous venous reflux was present in 9/10 individuals with VEGFR3 mutations, including two carriers. CONCLUSION: We propose that VEGFR3 mutations in humans cause lymphoedema through a failure of tissue protein and fluid absorption. This is due to a profound functional failure of initial lymphatics and is not explained by microlymphatic hypoplasia alone. The superficial venous valve reflux indicates the dual role of VEGFR-3 in lymphatic and venous development.

Recent advances in breast cancer-related lymphedema of the arm: lymphatic pump failure and predisposing factors.

Axillary surgery for breast cancer may be followed, months to years later, by chronic arm lymphedema. A simple 'stopcock' mechanism (reduced lymph drainage from the entire limb through surviving lymphatics) does not explain many clinical aspects, including the delayed onset and selective sparing of some regions, e.g., hand. Quantitative lymphoscintigraphy reveals that lymph drainage is slowed in the subcutis, where most of the edema lies, and in the subfascial muscle compartment, which normally has much higher lymph flows than the subcutis. Although the muscle does not swell significantly, the impaired muscle drainage correlates with the severity of arm swelling, indicating a likely key role for muscle lymphatic function. A new method, lymphatic congestion lymphoscintigraphy, showed that the edema is associated with a reduced contractility of the arm lymphatics; the weaker the active lymphatic pump, the greater the swelling. Delayed lymphatic pump failure may result from chronic raised afterload, as in hypertensive cardiac failure, and may account for the delayed onset of swelling. A further novel finding is that lymph flow is raised in both the subcutis and muscle of both arms in postsurgical breast patients who later developed breast cancer-related lymphedema (BCRL), compared with patients who did not develop BCRL. This new observation indicates a predisposition to BCRL in some women. Further evidence for predisposing abnormalities is the finding of lymphatic abnormalities in the contralateral (nonswollen) arm in women with established BCRL. Such predisposing factors could explain why some women develop BCRL after sentinel node biopsy, whereas others do not after clearance surgery. Future research must focus on prospective observations made from before surgery until BCRL develops.

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.

A quantitative lymphoscintigraphic evaluation of lymphatic function in the swollen hands of women with lymphoedema following breast cancer treatment.

In BCRL (breast cancer-related lymphoedema), arm swelling is unevenly distributed and some regions are partly or entirely spared. In particular, the hand may or not be swollen, but when involved functional impairment can be substantial. We have found previously that, when the ipsilateral hand is spared of swelling (in a limb with swelling proximal to the hand), the local lymph drainage rate constant (k) is at least as high as in the contralateral hand, contrary to the traditional 'stopcock' concept of reduced lymph drainage from the whole limb. In the light of this finding, we have investigated lymph drainage in the hands of eight women with BCRL and moderate-to-severe hand swelling, using gamma-camera quantitative lymphoscintigraphy. Images showed pronounced superficial activity in the ipsilateral swollen arms of most patients, indicating dermal backflow. k for 99mTc-labelled hIgG (human IgG) measured over 5 h in the subcutis of the ipsilateral swollen hand was 34+/-24% less than in the contralateral hand (P=0.013). Activity measured in the ipsilateral swollen forearm increased progressively, but there was very little increase in the contralateral forearm, indicating retention of 99mTc-labelled hIgG in the swollen forearm. It is concluded that lymphatic function in the swollen hand is impaired, and that there appears to be two populations of women with BCRL, i.e. spared-hand and swollen-hand, irrespective of the cancer treatment received.

Regional distribution of epifascial swelling and epifascial lymph drainage rate constants in breast cancer-related lymphedema.

BACKGROUND: The view that breast cancer-related lymphedema (BCRL) is a simple, direct mechanical result of axillary lymphatic obstruction ('stopcock' mechanism) appears incomplete, because parts of the swollen limb (e.g., hand) can remain nonswollen. The lymph drainage rate constant (k) falls in the swollen forearm but not in the spared hand, indicating regional differences in lymphatic function. Here the generality of the hypothesis that regional epifascial lymphatic failure underlies regional swelling was tested. To do so, the regional distribution of epifascial swelling along the forearm was compared with that of epifascial (subcutis) k. METHODS AND RESULTS: Epifascial k (local lymph flow per unit distribution volume) was measured by quantitative lymphoscintigraphy of subcutaneous radiolabeled human immunoglobulin IgG in regions of maximal and minimal % swelling in the ipsilateral swollen forearm, and at matching sites in the contralateral nonswollen arm, in 11 women with BCRL. Swelling was maximal distally in 5 patients and proximally in 6. Proximal k, -0.085 +/- 0.025% min(-1) (mean +/- SD), was 27% bigger than distal k, -0.067 +/- 0.021% min(-1), irrespective of swelling (p = 0.02, two-way repeated measures ANOVA). k fell by 11% from -0.080 +/- 0.028% min(-1) in the nonswollen arm to -0.072 +/- 0.021% min(-1) in the swollen arm (p = 0.17, t test). Local epifascial k was not significantly lower, however, at sites of maximal swelling than minimal swelling, and k correlated positively with arm circumference. CONCLUSIONS: A systematic difference in lymph drainage along the axis of the forearm was demonstrated for the first time. Local differences in epifascial k did not, however, explain the regionality of swelling, in keeping with previous evidence that epifascial k does not correlate with differences in swelling between arms, whereas subfascial k does. The results lead to the rejection of the hypothesis that epifascial (cf. subfascial) lymph drainage rate constants govern epifascial swelling in human forearm.

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

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.

Expansion of microvascular bed and increased solute flux in human Basal cell carcinoma in vivo, measured by fluorescein video angiography.

Human basal cell carcinoma (BCC) offers a unique opportunity to assess directly the microvascular abnormalities in a human cancer in vivo. Our objectives were to assess angiogenesis, perfusion, and changes in small solute exchange kinetics. The microcirculation of BCC and a normal (control) skin site was studied in 15 patients by laser Doppler fluximetry and videoangiography after rapid i.v. fluorescein injection. Microvascular morphometry was analyzed off line. Sodium fluorescein accumulation/clearance was recorded for 30 min, and fluorescence intensity (FI) was quantified by computer analysis of videotape image gray levels. In BCCs, the microvascular area fraction was 2.6-fold greater, microvessel length density 2.0-fold greater, average vessel image width 2.1-fold greater, and red cell flux 3.9-fold greater than in control sites (P < 0.01, paired t tests). The initial rate of rise of FI over 10 s was approximately 3-fold greater in BCC than control and correlated with vascular area fraction and red cell flux. Tissue then equilibrated faster in BCC, rate constant -(13.0 +/- 5.6) x 10(-3) s(-1) (mean +/- SD), than controls -(5.3 +/- 1.7) x 10(-3) s(-1), and plasma clearance was 2.6-fold higher in BCC than controls (P < 0.01, paired t test). Similarly, the rate constant of the subsequent clearance phase was approximately 2-fold greater in BCC, -(0.53 +/- 0.19) x 10(-3) s(-1), than controls, -(0.27 +/- 0.22) x 10(-3) s(-1) (P < 0.01). Removal rate constants were an order of magnitude slower than accumulation rate constants. The results demonstrate angiogenesis, increased perfusion, and a more rapid exchange of small solute in human BCC. FI itself is rejected as an index of permeability to small solutes (cf. 29) because it depends also on blood flow, endothelial area, microvascular volume, and interstitial fluid volume.