Polyester vascular patches acquire arterial or venous identity depending on their environment.

Polyester is commonly used in vascular surgery for patch angioplasty and grafts. We hypothesized that polyester patches heal by infiltration of arterial or venous progenitor cells depending on the site of implantation. Polyester patches were implanted into the Wistar rat aorta or inferior vena cava and explanted on day 7 or 30. Neointima that formed on polyester patches was thicker in the venous environment compared to the amount that formed on patches in the arterial environment. Venous patches had more cell proliferation and greater numbers of VCAM-positive and CD68-positive cells, whereas arterial patches had greater numbers of vimentin-positive and alpha-actin-positive cells. Although there were similar numbers of endothelial progenitor cells in the neointimal endothelium, cells in the arterial patch were Ephrin-B2- and notch-4-positive while those in the venous patch were Eph-B4- and COUP-TFII-positive. Venous patches treated with an arteriovenous fistula had decreased neointimal thickness; neointimal endothelial cells expressed Ephrin-B2 and notch-4 in addition to Eph-B4 and COUP-TFII. Polyester patches in the venous environment acquire venous identity, whereas patches in the arterial environment acquire arterial identity; patches in the fistula environment acquire dual arterial-venous identity. These data suggest that synthetic patches heal by acquisition of identity of their environment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3422-3431, 2017.

The ADP antagonist MRS2179 regulates the phenotype of smooth muscle cells to limit intimal hyperplasia.

PURPOSE: ADP plays an important part in platelet aggregation by activating P2Y1 and P2Y12 receptors. The ADP antagonist MRS2179 has been used in thrombosis-related treatments but its effects on vein graft (VG) remodeling is undefined. We examined the effect of MRS2179 on VG intimal hyperplasia and explored the mechanism of action. METHODS: A mouse model of VG transplantation was established. Mice underwent surgery and received MRS2179 by intraperitoneal injection every other day for 3 weeks. VG remodeling was assessed 4-weeks later. Vascular smooth muscle cells (VSMCs) were isolated and treated with MRS2179. The effect of MRS2179 on the proliferation, migration and inflammatory-cytokine expression of VSMCs was also evaluated. RESULTS: MRS2179 significantly inhibited VSMC proliferation compared with the control group. Significant inhibitory effects of MRS2179 on VSMC migration was observed in two-dimensional and three-dimensional models. The extent of intimal hyperplasia was significantly less in MRS2179 treated mice than in controls. Reduced migration of macrophage was found in MRS2179 treated mice. Expression of the inflammatory cytokines IL-1ß and TNF-α was decreased significantly in the MRS2179 treated group. In addition, decreased phosphorylation was found on Akt, Erk1/2 and p38. CONCLUSIONS: These data demonstrate that MRS2179 inhibits neointima formation in VGs by regulating the proliferation, and migration of VSMCs, macrophage migration, inflammatory-cytokine secretion and related signaling pathway. Our study provides novel insights regarding purinergic signaling in SMCs in vivo. The P2Y1 receptor may serve as a therapeutic target in neointima formation.

Morphofunctional characterization of decellularized vena cava as tissue engineering scaffolds.

Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2h and DS 2% for 1h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.

Implantation of inferior vena cava interposition graft in mouse model.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with ε-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (>90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

Tetrahydrobiopterin determines vascular remodeling through enhanced endothelial cell survival and regeneration.

BACKGROUND: Endothelial cell (EC) survival and regeneration are important determinants of the response to vascular injury that leads to neointimal hyperplasia and accelerated atherosclerosis. Nitric oxide (NO) is a key regulator of EC and endothelial progenitor cell function, but the pathophysiological mechanisms that regulate endothelial NO synthase in endothelial regeneration remain unclear. METHODS AND RESULTS: Endothelium-targeted overexpression of GTP cyclohydrolase (GCH) I increased levels of the endothelial NO synthase cofactor, tetrahydrobiopterin, in an EC-specific manner and reduced neointimal hyperplasia in experimental vein grafts in GCH/apolipoprotein E-knockout mice. These effects were mediated through enhanced donor-derived survival and recipient-derived repopulation of GCH transgenic ECs, revealed by tracking studies in Tie2-LacZ/GCH-Tg/apolipoprotein E-knockout recipient mice or donor grafts, respectively. Endothelial GCH overexpression increased endothelial NO synthase coupling and enhanced the proliferative capacity of ECs and circulating endothelial progenitor cell numbers after vascular injury. CONCLUSIONS: These observations indicate that endothelial tetrahydrobiopterin availability modulates neointimal hyperplasia after vascular injury via accelerated EC repopulation and growth. Targeting tetrahydrobiopterin-dependent endothelial NO synthase regulation in the endothelium is a rational therapeutic target to enhance endothelial regeneration and reduce neointimal hyperplasia in vascular injury states.

Comparison of vascular smooth muscle cells in canine great vessels.

BACKGROUND: Elucidating the histological characteristics of normal vascular smooth muscle cells (VSMCs) is important for understanding mechanisms of development, disease etiology and the remodeling and/or regeneration process of the vessel. However, knowledge regarding VSMCs is focused primarily on the artery. Although the characteristics of each great vessel are documented, few studies have examined VSMCs in parallel within each great vessel. The present study focused on comparing characteristics of canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC), simultaneously. RESULTS: Western blot and immunohistochemistry were used to determine VSMC protein content for alpha smooth muscle actin (ASMA), calponin, myosin heavy chain (MHC) and its isozyme SM2, and non-muscle myosin heavy chain B (SMemb). Thickness and ratio of the VSMC layer were also measured. Expression levels of ASMA, calponin and SM2 significantly differed between vessels, except between mPA and either bPA, Ao and IVC vessels. Expression levels of MHC were significantly different in all vessels, whilst expression of SMemb was significantly different in the Ao compared with either bPA and mPA vessels. All vessels were significantly different with respect to total wall and VSMC layer thickness. The ratio between VSMC layer and total wall thickness was significantly different for each vessel, except between bPA and mPA vessels. Histological analysis of the IVC revealed that the VSMC layer does not line evenly and continuously through the long axis or transverse sections. With respect to the pulmonary artery, calponin was expressed to a greater extent in the mPA compared with the bPA (P < 0.01*). In contrast, MHC and SM2 were expressed to a greater extent in the bPA compared with the mPA (P < 0.01*). Differences in VSMC distribution indicate structural differences in the proximal and distal pulmonary artery bifurcation. CONCLUSION: Our results show that the VSMC expression pattern in each great vessel is unique and suggestive of the developmental differences between great vessels. We believe this study provides basic data for the pathology, etiology and regenerative capability of the vessels.

Agenesia da veia cava inferior / Agenesis of the inferior vena cava

Agenesia da veia cava inferior é uma malformação rara. Sua causa mais comum é a disgenesia durante a embriogênese, mas também pode estar relacionada a trombose intrauterina ou perinatal. Normalmente é assintomática, em associação, ou não, com outras malformações congênitas, e pode cursar com maior risco de insuficiência venosa crônica e trombose venosa profunda, especialmente em jovens. Seu diagnóstico frequentemente é acidental, durante cirurgias abdominais ou procedimentos radiológicos. Relatamos cinco casos de agenesia da veia cava inferior detectada durante procedimentos eletrofisiológicos.

Agenesis of the inferior vena cava is a rare malformation. Its most common cause is dysgenesis during embryogenesis, but it may also be related to intrauterine or perinatal thrombosis. It is usually asymptomatic, associated or not with other congenital malformations and may be related to increased risk of chronic venous insufficiency and deep vein thrombosis, especially in young individuals. Diagnosis is often incidental, during abdominal surgery or radiological procedures. We reported five cases of agenesis of the inferior vena cava detected during electrophysiological procedures.

Lineage tree for the venous pole of the heart: clonal analysis clarifies controversial genealogy based on genetic tracing.

RATIONALE: Genetic tracing experiments and cell lineage analyses are complementary approaches that give information about the progenitor cells of a tissue. Approaches based on gene expression have led to conflicting views about the origin of the venous pole of the heart. Whereas the heart forms from 2 sources of progenitor cells, the first and second heart fields, genetic tracing has suggested a distinct origin for caval vein myocardium, from a proposed third heart field. OBJECTIVE: To determine the cell lineage history of the myocardium at the venous pole of the heart. METHODS AND RESULTS: We used retrospective clonal analyses to investigate lineage segregation for myocardium at the venous pole of the mouse heart, independent of gene expression. CONCLUSIONS: Our lineage analysis unequivocally shows that caval vein and atrial myocardium share a common origin and demonstrates a clonal relationship between the pulmonary vein and progenitors of the left venous pole. Clonal characteristics give insight into the development of the veins. Unexpectedly, we found a lineage relationship between the venous pole and part of the arterial pole, which is derived exclusively from the second heart field. Integration of results from genetic tracing into the lineage tree adds a further temporal dimension to this reconstruction of the history of venous myocardium and the arterial pole.

Determining the fate of seeded cells in venous tissue-engineered vascular grafts using serial MRI.

A major limitation of tissue engineering research is the lack of noninvasive monitoring techniques for observations of dynamic changes in single tissue-engineered constructs. We use cellular magnetic resonance imaging (MRI) to track the fate of cells seeded onto functional tissue-engineered vascular grafts (TEVGs) through serial imaging. After in vitro optimization, murine macrophages were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles and seeded onto scaffolds that were surgically implanted as inferior vena cava interposition grafts in SCID/bg mice. Serial MRI showed the transverse relaxation times (T(2)) were significantly lower immediately following implantation of USPIO-labeled scaffolds (T(2) = 44 ± 6.8 vs. 71 ± 10.2 ms) but increased rapidly at 2 h to values identical to control implants seeded with unlabeled macrophages (T(2) = 63 ± 12 vs. 63 ± 14 ms). This strongly indicates the rapid loss of seeded cells from the scaffolds, a finding verified using Prussian blue staining for iron containing macrophages on explanted TEVGs. Our results support a novel paradigm where seeded cells are rapidly lost from implanted scaffolds instead of developing into cells of the neovessel, as traditionally thought. Our findings confirm and validate this paradigm shift while demonstrating the first successful application of noninvasive MRI for serial study of cellular-level processes in tissue engineering.

Formation of the azygos vein / Formación de la vena ácigos

The aim of the present study was to determine the most common origin of the azygos vein. Thirty cadavers male and female, white and non-white adult individuals of different ages fixed in 10 percent formaldehyde and dissected. All cadavers had an undisclosed clinical death and were donated to the Universidade Estadual de Ciências da Saúde de Alagoa s, Brazil. Eleven different formations were found. The right subcostal vein was was only observed in 13 cases (43.33 percent); the azygos vein was formed by the confluence of the right subcostal and right ascending lumbar vein in three cases (10 percent); by the right subcostal vein with a contribution from the inferior vena cava (IVC) in three cases (10 percent); by the right subcostal with contribution from IVC and right ascending lumbar vein in three cases (10 percent); by the right and left subcostal veins in two cases (6.66 percent); by the right and left subcostal veins and contribution from the IVC in one case (3.33 percent); by the right and left subcostal veins and left accessory renal vein in one case (3.33 percent); by the left renal vein in one case (3.33 percent); by the right subcostal and left gonadal veins with contribution from the IVC in one case (3.33 percent); by the right subcostal and left renal veins in one case (3.33 percent); and composed by the continuation of the 11th posterior intercostal vein in one case (3.33 percent). Based on the results, the right subcostal vein was the only structure with a significant presence in the formation of the azygos vein.

El objetivo del estudio fue verificar cual es la disposición más frecuente del origen de la vena ácigos. Fueron disecados 30 cadáveres de individuos adultos, de ambos sexos, de diferentes grupos étnicos, fijados en formaldehído al 10 por ciento, donados a la Universidade Estadual de Ciencias da Saúde de Alagoas. Se encontraron 11 formaciones diferentes. En 13 casos (43,33 por ciento) se observó sólo la vena subcostal derecha; en 3 casos (10 por ciento) la vena ácigos estaba formada por la confluencia de las venas subcostal derecha y lumbar ascendente derecha; en 3 casos (10 por ciento) formado por las venas subcostal derecha y una contribución de la vena cava inferior VCI; en 3 casos (10 por ciento) por las venas subcostal derecha y contribución de la VCI y lumbar ascendente derecha; 2 casos (6,66 por ciento) por las venas subcostales derecha e izquierda; en 1 caso (3,33 por ciento) por las venas subcostal derecha, izquierda y contribución de la VCI; en 1 caso (3,33 por ciento) por las venas subcostal derecha e izquierda y renal accesoria izquierda; en 1 caso (3,33 por ciento) por la vena renal izquierda; en1 caso (3,33 por ciento) por las venas subcostal derecha, gonadal izquierda y contribución de la VCI; en 1 caso (3,33 por ciento) por las venas subcostal derecha y renal izquierda y en 1 caso (3,33 por ciento) por la continuación de la 11 vena intercostal posterior. Con base en los resultados podemos concluir que la vena subcostal derecha fue la única estructura con presencia significativa en la formación de la vena ácigos.

Modulation of vein function by perivascular adipose tissue.

Although a number of studies have shown that perivascular adipose tissue (PVAT) attenuates arterial contraction through the release of perivascular-derived relaxation factors (PVRF), the role of PVAT in modulating venous function and its mechanism(s) remained unknown. Here we examined the role of PVAT in the modulation of vascular function in the inferior vena cava. Venous rings from male Wistar rats were prepared with both endothelium and PVAT intact, with either PVAT or endothelium removed, or with both endothelium and PVAT removed for functional studies. Contractile response to phenylephrine, U 46619, or 5-hydroxytryptamine was significantly attenuated in PVAT+ as compared with PVAT- veins. PVAT- vessels with intact endothelium (E+) pre-contracted with phenylephrine showed a concentration-dependent relaxation response to angiotensin 1-7 [Ang-(1-7)], and this response was abolished by the removal of endothelium, and by Ang-(1-7) (Mas) receptor antagonists D-Ala-Ang-(1-7) (A779) or D-Pro(7)-Ang-(1-7). Donor solution incubated with a PVAT+ ring induced a relaxation response in the E+ recipient vessel but not in E- recipient vessel. The use of specific channel blockers and enzyme inhibitors showed that Ang-(1-7) is a transferable PVRF that induces endothelium-dependent relaxation through NO release and activation of voltage-dependent potassium (K(+)) channels (K(v)) channels. We conclude that venous PVAT attenuates agonist-induced contraction by releasing Ang-(1-7), which causes relaxation of smooth muscle through endothelial NO release and activation of K(v) channels.

Tissue-engineered vascular grafts: does cell seeding matter?

PURPOSE: Use of tissue-engineered vascular grafts (TEVGs) in the repair of congenital heart defects provides growth and remodeling potential. Little is known about the mechanisms involved in neovessel formation. We sought to define the role of seeded monocytes derived from bone marrow mononuclear cells (BM-MNCs) on neovessel formation. METHODS: Small diameter biodegradable tubular scaffolds were constructed. Scaffolds were seeded with the entire population of BM-MNC (n = 15), BM-MNC excluding monocytes (n = 15), or only monocytes (n = 15) and implanted as infrarenal inferior vena cava (IVC) interposition grafts into severe combined immunodeficiency/bg mice. Grafts were evaluated at 1 week, 10 weeks, or 6 months via ultrasonography and microcomputed tomography, as well as by histologic and immunohistochemical techniques. RESULTS: All grafts remained patent without stenosis or aneurysm formation. Neovessels contained a luminal endothelial lining surrounded by concentric smooth muscle cell layer and collagen similar to that seen in the native mouse IVC. Graft diameters differed significantly between those scaffolds seeded with only monocytes (1.022 +/- 0.155 mm) and those seeded without monocytes (0.771 +/- 0.121 mm; P = .021) at 6 months. CONCLUSIONS: Monocytes may play a role in maintaining graft patency. Incorporation of such findings into the development of second-generation TEVGs will promote graft patency and success.

Regeneration of the inferior vena cava with a bioabsorbable polymer implant: a histological study.

BACKGROUND: Cell implantation into ischemic regions has recently been introduced as a novel strategy for therapeutic angiogenesis. Little is known, however, about the process of blood vessel regeneration, particularly that of the inferior vena cava (IVC). The indicators of normal angiogenesis are also unestablished. PURPOSE: To investigate the process of regeneration of the IVC from a histological viewpoint and to speculate on how the new formation and regeneration of the blood vessels proceed. MATERIALS AND METHODS: Our previous studies showed that a bioabsorbable polymer patch implanted into the IVC formed vessels resembling the native IVC (J Gastrointest Surg 2005;9:789). Using this model system, we investigated the histology and time course of IVC regeneration in the graft site. A 3 x 2 portion of infrahepatic IVC was substituted by a bioabsorbable polymer patch of the same size in hybrid pigs. The patched area was excised for histology at 2 weeks and 3, 6, and 12 months after implantation (n = 3, each). RESULTS: By 2 weeks, the patched area had developed vascular endothelial cells of the same type seen in native veins. The polymer implant was still detectable at 2 weeks but histologically absorbed at 3 months. Smooth muscle was barely formed at 2 weeks, but the ratio of smooth muscle to subendothelial connective tissue gradually increased as time advanced to 3, 6, and 12 months. Even at the last observation at 12 months, however, the amount of smooth muscle formed made up no more than one-half of the native IVC. The case with the elastic fibers accounted for about 90% of the total number of native fibers at 12 months. On gross examination, the patched area resembled the native IVC at 3 months after implantation. CONCLUSION: These results demonstrated that the subendothelial tissue regenerated gradually, requiring more than 1 year to resemble native tissue, whereas the vascular endothelium regenerated in the early phase after injury. Our findings make it possible to establish criteria by which to evaluate venous regeneration.

Tissue engineered venous matrices for potential applications in the urogenital tract.

Tissue engineering is lacking inexpensive, easily applicable techniques for tissue replacement. We investigated the potential use of native veins for tissue-engineering applications in the urological field. Forty-eight porcine veins, half seeded with urothelial cells and half unseeded, were kept in vitro for 7 days. Four seeded and four unseeded scaffolds were analyzed after 3 and 7 days. The remaining 32 veins were implanted subcutaneously into 16 athymic mice. Four athymic mice were sacrificed after 2, 4, 8, and 12 weeks. Histochemistry, immunohistochemistry (anti-pancytokeratin AE1/AE3, anti-desmin), western blot analyses (CD31), and scanning electron microscopy were performed in the retrieved specimens. The histochemistry of the seeded matrices showed the presence of urothelial cells in vitro and in vivo. After 12 weeks, a multilayer of urothelial cells was present in the hemotoxylin and eosin staining, positive for anti-pancytokeratin AE1/AE3. The western blot analyses showed vascularization of the veins in vivo. The results of scanning electron microscopy revealed a cellular layer on the veins. Native venous matrices may be used as tissue-engineered constructs for reconstructing the urinary tract. The clinical relevance of this approach must be proven in a large-animal model.

Development of a spontaneously beating vein by cardiomyocyte transplantation in the wall of the inferior vena cava in a rat: a pilot study.

PURPOSE: This study was conducted to determine whether it is feasible to develop a vein that rhythmically beats by implanting immature cardiomyocytes in its wall. METHODS: Neonatal cardiomyocytes (5 x 10(6) cells each) were transplanted into the wall of the inferior vena cava in six female Fischer rats; in six rats, only the medium was transplanted. At 3 weeks after transplantation, the grafted site of the inferior vena cava was exposed and videotaped, and then processed for histology. RESULTS: Distinct rhythmic beating of the vena cava at the site of cell injection (at a rate lower than aortic beating) was observed in all six rats treated with neonatal cardiomyocyte injections, but in none of the six that received the medium. The vena cava continued to beat spontaneously and rhythmically after the aortas were clamped and after the heart was excised. The beating was manifest by visual contraction and relaxation of the vessel wall. The spontaneous beating rate was 101 +/- 7 beats/min at 1 to 3 minutes after excision of the heart. Hematoxylin and eosin staining showed viable grafts in the wall of the vena cava in all that were implanted with neonatal cardiac cells; but in none of the vena cava that received the medium. Neonatal cardiomyocytes in the graft matured with cross striations and stained positive for the muscle marker sarcomeric actin. CONCLUSIONS: The present study demonstrates that neonatal cardiomyocytes survive, mature, and spontaneously and rhythmically contract when implanted in the wall of a vein.

Transport in rat vessel walls. II. Macromolecular leakage and focal spot size growth in rat arteries and veins.

Transendothelial lipid transport into and spread in the subendothelial intima of large arteries, and subsequent lipid accumulation, appear to start plaque formation. We experimentally examine transendothelial horseradish peroxidase (HRP) transport in vessels that are usually, e.g., pulmonary artery (PA), or almost always, e.g., inferior vena cava (IVC), atherosclerosis resistant vs. disease prone, e.g., aorta, vessels. In these vessels, HRP traverses the endothelium at isolated, focal spots, rather than uniformly, for short circulation times. For femoral vein HRP introduction, PA spots have 30-s radii [ approximately 53.2 microm (SD 10.4); compare aorta: 54.6 microm (SD 8.75)] and grow quickly from 30 s to 1 min (40%, P<0.05) and more slowly afterward (P>0.05). This trend resembles the aorta, suggesting the PA has a similarly sparse intima. With carotid artery (CA) HRP introduction, the 30-s spot (132.86 +/- 37.32 microm) is far larger than the PAs, grows little ( approximately 28%, P<0.05) from 30 to 60 s, and is much flatter than the artery curves. Transverse electron microscopic sections after approximately 10 min HRP circulation show thin, intense staining immediately beneath both vessels' endothelia with an almost step change to diffuse staining beyond. This indicates the existence of a sparse, subendothelial intima, even when there is no internal elastic lamina (IVC). This motivates a simple model that translates growth rates into lower bounds for the flow through focal leaks. The model results and our earlier wall and medial hydraulic conductivity data explain these spot growth curves and point to differences in transport patterns that might be relevant in understanding the immunity of IVC to disease initiation.

Coculture of endothelial and smooth muscle cells on a collagen membrane in the development of a small-diameter vascular graft.

In this study, we have evaluated the feasibility of developing a biodegradable collagenous small diameter vascular graft of 2mm diameter and 1cm length. In brief, bi-layer type I collagen membrane was fabricated under vacuum suction and lyophilization methods. The smooth muscle cells were inoculated into the lower side of the porous membrane, while endothelial cells were seeded onto upper smooth side of the membrane. After cultured for 7 days, the vascular substitute was either harvested for in vitro examination or in vivo implanted in the subcutaneous layer for biocompatibility test. The tubular vascular prosthesis was then used as a temporary absorbable guide that served as an in vivo vascular graft to promote the complete regeneration of rat inferior vena cava. After implantation for 12 weeks, a thin continuous layer of endothelial cells and smooth muscle cells were lined with the vascular lumen and tunic media, respectively. Histology results showed that there were no signs of significant thrombogeneity and intima hyperplasia. This tissue engineered vascular substitute not only had enough tensile strength and good biocompatibility, but also advanced vascular regeneration. In the future, we suggest that this biodegradable vascular substitute will provide with the possibility in application on small diameter prosthetic grafts in artificial blood vessels.

Preliminary experience with tissue engineering of a venous vascular patch by using bone marrow-derived cells and a hybrid biodegradable polymer scaffold.

OBJECTIVE: Currently available synthetic polymer vascular patches used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. These problems may be avoided by vascular patches tissue-engineered with autologous stem cells and biodegradable polymeric materials. The objective of this study was to develop a tissue-engineered vascular patch by using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. METHODS: Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. Tissue-engineered vascular patches (15 mm wide x 30 mm long) were fabricated by seeding vascular cells onto PGA/PLCL scaffolds and implanted into the inferior vena cava of bone marrow donor dogs. RESULTS: Compared with PLCL scaffolds, PGA/PLCL scaffolds exhibited tensile mechanical properties more similar to those of dog inferior vena cava. Eight weeks after implantation of vascular patches tissue-engineered with BMCs and PGA/PLCL scaffolds, the vascular patches remained patent with no sign of thrombosis, stenosis, or dilatation. Histological, immunohistochemical, and scanning electron microscopic analyses of the retrieved vascular patches revealed regeneration of endothelium and smooth muscle, as well as the presence of collagen. Calcium deposition on tissue-engineered vascular patches was not significantly different from that on native blood vessels. Immunofluorescent double staining confirmed that implanted BMCs survived after implantation and contributed to regeneration of endothelium and vascular smooth muscle in the implanted vascular patches. CONCLUSIONS: This study demonstrates that vascular patches can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats.

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33 degrees C. A temperature shift to 37 degrees C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.

Evaluation of tissue-engineered vascular autografts.

This study evaluated the endothelial function and mechanical properties of tissue-engineered vascular autografts (TEVAs) constructed with autologous mononuclear bone marrow cells (MN-BMCs) and a biodegradable scaffold using a canine inferior vena cava (IVC) model. MN-BMCs were obtained from a dog and seeded onto a biodegradable tubular scaffold consisting of polyglycolide fiber and poly(L-lactide-co-epsilon-caprolactone) sponge. This scaffold was implanted in the IVC of the same dog on the day of surgery. TEVAs were analyzed biochemically, biomechanically, and histologically after implantation. When TEVAs were explanted and stimulated with acetylcholine at 1 month, they produced nitrates and nitrites dose dependently. N(G)-nitro-L-arginine methylester significantly inhibited these reactions. With stimulation by acetylcholine, factor VIII-positive cells of TEVAs produced endothelial nitric oxide synthase proteins, and the ratio of endothelial nitric oxide synthase/s17 mRNA was similar among native IVC and TEVAs 1 and 3 months after implantation. TEVAs had biochemical properties and wall thickness similar to those of native IVC at 6 months after implantation, and tolerated venous pressure well without any problems such as calcification. The number of inflammatory cells in TEVAs and the ratio of CD4/s17 mRNA decreased significantly with time. These results indicate that TEVAs are a biocompatible material with functional endothelial cells and biomechanical properties and do not have unwanted side effects.