Antigen removal process preserves function of small diameter venous valved conduits, whereas SDS-decellularization results in significant valvular insufficiency.

Chronic venous disease (CVD) is the most common reported chronic condition in the United States, affecting more than 25 million Americans. Regardless of its high occurrence, current therapeutic options are far from ideal due to their palliative nature. For best treatment outcomes, challenging cases of chronic venous insufficiency (CVI) are treated by repair or replacement of venous valves. Regrettably, the success of venous valve transplant is dependent on the availability of autologous venous valves and hindered by the possibility of donor site complications and increased patient morbidity. Therefore, the use of alternative tissue sources to provide off-the-shelf venous valve replacements has potential to be extremely beneficial to the field of CVI. This manuscript demonstrates the capability of producing off-the-shelf fully functional venous valved extracellular matrix (ECM) scaffold conduits from bovine saphenous vein (SV), using an antigen removal (AR) method. AR ECM scaffolds maintained native SV structure-function relationships and associated venous valves function. Conversely, SDS decellularization caused significant changes to the collagen and elastin macromolecular structures, resulting in collagen fibril merging, elimination of fibril crimp, amalgaming collagen fibers and fragmentation of the inner elastic lamina. ECM changes induced by SDS decellularization resulted in significant venous valve dysfunction. Venous valved conduits generated using the AR approach have potential to serve as off-the-shelf venous valve replacements for CVI. STATEMENT OF SIGNIFICANCE: Retention of the structure and composition of extracellular matrix (ECM) proteins within xenogeneic scaffolds for tissue engineering is of crucial importance, due to the undeniable effect ECM proteins can impose on repopulating cells and function of the resultant biomaterial. This manuscript demonstrates that alteration or elimination of ECM proteins via commonly utilized decellularization approach results in complete disruption of venous valve function. Conversely, retention of the delicate ECM structure and composition of native venous tissue, using an antigen removal tissue processing method, results in preservation of native venous valve function.

Correlative Fluorescence and Scanning Electron Microscopy to Study Lymphovenous Valve Development.

Lymph collected from throughout the body is exclusively returned to blood circulation via two pairs of bilaterally located lymphovenous valves. Lymphovenous valves share numerous similarities with lymphatic and venous valves and are defective in multiple mouse models of lymphedema or lymphatic dysfunction. Here we describe a protocol that combines the strengths of fluorescence microscopy and scanning electron microscopy to precisely locate and analyze the topography of developing lymphovenous valves at high resolution.

The influence of age on valve disease in patients with varicose veins analysed by transmission electron microscopy and stereology.

BACKGROUND: The aim of this study was to investigate the influence of age on the ultrastructure of venous valve morphology in patients with C2 classified chronic venous disorders according to the CEAP classification. PATIENTS AND METHODS: The study population consisted of 16 consecutive patients with varicose veins (C2). The mean age was 49.8 years (30-66). The (pre-) terminal valve including the vessel wall was harvested within the proximal 2 centimetres of the great saphenous vein. The mean thickness (volume-to-surface ratio = V/S ratio) of elastin, collagen, endothelium and of the entire valve was determined. A blinded morphologist performed the examination by transmission electron microscopy and stereology. Analyses by Pearson's product moment correlation, Kendall's tau and Spearman's rank correlation were performed to investigate whether there is a correlation between age and the ultrastructural morphology. RESULTS: Stereological analysis of the valves demonstrated a mean V/S ratio (signifying a thickness estimation) for elastin of 0.87 µm3/µm2, for collagen of 18.0 µm3/µm2, for endothelium of 0.65 µm3/µm2, and for the entire valve of 25.2 µm³/µm². Statistical analyses showed no statistically significant correlation between age and the ultrastructural morphology in this patient group. CONCLUSIONS: The ultrastructural morphology of the venous valves in chronic venous disorders may not depend on age in patients presenting with C2 disease. This conclusion may or may not apply to all C classes as we investigated a homogenous group of patients with C2 limbs.

Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.

Valve disease in chronic venous disorders: a quantitative ultrastructural analysis by transmission electron microscopy and stereology.

INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 µm3/m2; in group B, 0.67 µm3/m2; in group C, 0.97 µm3/m2. The ratio was similar for collagen (A, 15.7 µm3/m2; B, 26.8 µm3/m2; C, 30.1 µm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Ophthalmic and facial veins are not valveless.

BACKGROUND: The ophthalmic and facial veins are frequently stated to be devoid of valves, facilitating the spread of infection from the mid-face to the cavernous sinus. METHODS: Twelve superior and eight inferior ophthalmic veins together with 13 angular and facial veins were harvested from adult cadavers. Each vein was opened longitudinally and examined by stereomicroscopy; the number, location and geometry of valve cusps were recorded. RESULTS: Ten valves were identified in nine (75%) superior ophthalmic vein specimens: four valves were in the superior ophthalmic vein and the remainder were located near its origin from angular and supra-orbital tributaries. No valves were seen in the inferior ophthalmic vein. Seventeen bicuspid valves were identified in tributaries of the angular vein or in the facial vein, but none were in the angular vein itself. Four of seven facial vein segments extending to the lower border of the mandible had valves. The orientation of valve cusps predicted the following blood flow: in the facial vein, inferiorly; in the superior ophthalmic vein, towards the cavernous sinus; and in the angular vein, to the facial or superior ophthalmic vein. CONCLUSIONS: This study demonstrates, for the first time, the existence of valves in the superior ophthalmic vein and its two main tributaries. Valves were also seen in the facial vein. It is not the absence of venous valves but the existence of communications between the facial vein and cavernous sinus and the direction of blood flow that is important in the spread of infection from the face.