The allostery and modification of hGHRH molecules and specific dimer produced significant fertility effect by proliferating and activating in-situ ovarian mesenchymal stem cells.

The negative coordination of growth hormone secretagogue receptor (GHS-R) and growth hormone-releasing hormone receptor (GHRH-R) involves in the repair processes of cellular injury. The allosteric U- or H-like modified GHRH dimer Grinodin and 2Y were comparatively evaluated in normal Kunming mice and hamster infertility models induced by CPA treatment. 1-3-9 µg of Grinodin or 2Y per hamster stem-cell-exhaustion model was subcutaneously administered once a week, respectively inducing 75-69-46 or 45-13-50 % of birth rates. In comparison, the similar mole of human menopausal gonadotropin (hMG) or human growth hormone (hGH) was administered once a day but caused just 25 or 20 % of birth rates. Grinodin induced more big ovarian follicles and corpora lutea than 2Y, hMG, hGH. The hMG-treated group was observed many distorted interstitial cells and more connective tissues and the hGH-treated group had few ovarian follicles. 2Y had a plasma lifetime of 21 days and higher GH release in mice, inducing lower birth rate and stronger individual specificity in reproduction as well as only promoting the proliferation of mesenchymal-stem-cells (MSCs) in the models. In comparison, Grinodin had a plasma lifetime of 30 days and much lower GH release in mice. It significantly promoted the proliferation and activation of ovarian MSCs together with the development of follicles in the models by increasing Ki67 and GHS-R expressions, and decreasing GHRH-R expression in a dose-dependent manner. However, the high GH and excessive estrogen levels in the models showed a dose-dependent reduction in fertility. Therefore, unlike 2Y, the low dose of Grinodin specifically shows low GHS-R and high GHRH-R expressions thus evades GH and estrogen release and improves functions of organs, resulting in an increase of fertility.

Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke.

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown. Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model. Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions. Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke. Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

Bone Marrow Stromal Cells Combined With Sodium Ferulate and n-Butylidenephthalide Promote the Effect of Therapeutic Angiogenesis via Advancing Astrocyte-Derived Trophic Factors After Ischemic Stroke.

Being a potential candidate for stroke treatment, bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) have been demonstrated to be able to enhance angiogenesis and proliferation of reactive astrocytes, which subsequently leads to the amelioration of neurological injury. Increasing evidence further indicates that combining BM-MSCs with certain agents, such as simvastatin, may improve therapeutic effects. Sodium ferulate (SF) and n-butylidenephthalide (BP), two main components of Radix Angelica Sinensis, are proven to be important regulators of stem cells in cell migration, differentiation, and pluripotency maintenance. This study aimed to investigate whether combining BM-MSCs with SF and BP had better therapeutic effect in the treatment of stroke, and the underlying molecular basis for the therapeutic effects was also investigated. The results showed that combination treatment notably reduced neurological injury after stroke and increased the expression of astrocyte-derived vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and von Willebrand factor-positive vascular density in the ischemic boundary zone as evaluated by immunofluorescence staining. After treatment with BM-MSCs plus SF and BP, astrocytes showed increased expression of VEGF and BDNF by upregulating protein kinase B/mammalian target of rapamycin (AKT/mTOR) expression in an oxygen- and glucose-deprived (OGD) environment. Human umbilical vein endothelial cells (HUVECs) incubated with the conditioned medium (CM) derived from OGD astrocytes treated with BM-MSCs plus SF and BP showed significantly increased migration and tube formation compared with those incubated with the CM derived from OGD astrocytes treated with BM-MSCs alone. These results demonstrate that combination treatment enhances the expression of astrocyte-derived VEGF and BDNF, which contribute to angiogenesis after cerebral ischemia, and the underlying mechanism is associated with activation of the astrocytic AKT/mTOR signaling pathway. Our study provides a potential therapeutic approach for ischemic stroke.

Xyloketal B attenuates atherosclerotic plaque formation and endothelial dysfunction in apolipoprotein e deficient mice.

Our previous studies demonstrated that xyloketal B, a novel marine compound with a unique chemical structure, has strong antioxidant actions and can protect against endothelial injury in different cell types cultured in vitro and model organisms in vivo. The oxidative endothelial dysfunction and decrease in nitric oxide (NO) bioavailability are critical for the development of atherosclerotic lesion. We thus examined whether xyloketal B had an influence on the atherosclerotic plaque area in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet and investigated the underlying mechanisms. We found in our present study that the administration of xyloketal B dose-dependently decreased the atherosclerotic plaque area both in the aortic sinus and throughout the aorta in apoE-/- mice fed a high-fat diet. In addition, xyloketal B markedly reduced the levels of vascular oxidative stress, as well as improving the impaired endothelium integrity and NO-dependent aortic vasorelaxation in atherosclerotic mice. Moreover, xyloketal B significantly changed the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and Akt without altering the expression of total eNOS and Akt in cultured human umbilical vein endothelial cells (HUVECs). Here, it increased eNOS phosphorylation at the positive regulatory site of Ser-1177, while inhibiting phosphorylation at the negative regulatory site of Thr-495. Taken together, these findings indicate that xyloketal B has dramatic anti-atherosclerotic effects in vivo, which is partly due to its antioxidant features and/or improvement of endothelial function.

Vascular fibrosis in atherosclerosis.

Vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to excessive deposition of extracellular matrix (ECM), links with many clinical diseases and pathological progresses including atherosclerosis. It involves proliferation of vascular smooth muscle cell (VSMC), accumulation of ECM and inhibition of matrix degradation. The risk factors associated with cardiovascular disease, including hypertension, hyperglycemia, dyslipidemia and hyperhomocysteinemia (HHcy), are also suggested as initiation and progression factors of vascular fibrosis. Vascular fibrosis has been found to relate to renin-angiotensin-aldosterone system (RAAS), oxidative stress, inflammatory factors, growth factors and imbalance of endothelium-derived cytokine secretion. Angiotensin II (Ang II) and aldosterone, the circulating effector hormones of RAAS, are recognized as responsible for the pathophysiology of vascular fibrosis. Transforming growth factor-beta (TGF-beta) plays a critical role in ECM accumulation and vascular remodeling via up-regulating the production of several agents including connective tissue growth factor (CTGF) and fibroblast growth factor. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) results in collagen accumulation and adverse matrix remodeling. Aberrant expression or function of peroxisome proliferator-activated receptor gamma (PPAR gamma) is also associated with, and very likely contributes to, the progression of pathological fibrosis and vascular remodeling. In this review, we discuss the pathogenesis of vascular fibrosis in atherosclerosis with focus on the networking among main responsible mediators. The main pathophysiologic factors leading to vascular fibrosis will also be discussed.

Homocysteine-impaired angiogenesis is associated with VEGF/VEGFR inhibition.

This study investigated the effects of homocysteine (Hcy) on angiogenesis in cultured human umbilical vein endothelial cells (HUVEC) and zebrafish embryos. We found that Hcy (50 micromol/L) significantly decreased cell numbers, viability, and induced a G1/S arrest in HUVEC in the presence of adenosine (Ade, 50 micromol/L). Hcy, in combination with Ade, reduced migration and suppressed tube-like formation on Matrigel in HUVEC. Further, Hcy reduced subintestinal vessel formation in zebrafish embryos. Interestingly, Hcy-induced inhibitory effects on cell growth, migration, tube-like formation, and vessel formation in HUVEC and zebra fish embryos were abolished by the supplement of recombinant VEGF (10 ng/ml). Finally, Hcy in combination with Ade reduced the mRNA levels of VEGF, VEGFR-1, VEGFR-2, and attenuated protein levels of VEGF, ERK1/2 and Akt. The present study suggests that Hcy inhibits angiogenesis, and that the mechanism anti-angiogenic effects of Hcy may be through VEGF/VEGFR, Akt, and ERK1/2 inhibition.

Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition.

Hyperhomocysteinemia (HHcy), a risk factor for cardiovascular disease, is associated with endothelial dysfunction. Ginsenoside Rb1, the major active constituent of ginseng, potently attenuates homocysteine (Hcy)-induced endothelial damage. However, the underlying mechanism remains unknown. In this study, we have investigated the effect of Ginsenoside Rb1 on Hcy-induced endothelial dysfunction and its underlying signal pathway in vivo and in vitro. Ginsenosides prevented Hcy-induced impairment of endothelium-dependent relaxation and Rb1 reversed Hcy-induced reduction of NO production in a dose-dependent manner as detected by nitrate reductase method. Rb1 activated serine-1177 phosphorylation of endothelial nitric oxide synthase (eNOS) and serine-473 phosphorylation of Akt, while inhibited threonine-495 phosphorylation of eNOS as detected by western blotting. Rb1-induced phosphorylation of serine-1177 was significantly inhibited by wortmannin, PI3K inhibitor or SH-5, an Akt inhibitor, and partially reversed by Phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA also stimulated phosphorylation of threonine-495 which was inhibited by Rb1. Here we show for the first time that Rb1 prevents Hcy-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.

Homocysteine impaired endothelial function through compromised vascular endothelial growth factor/Akt/endothelial nitric oxide synthase signalling.

1. Hyperhomocysteinaemia (HHcy) is associated with endothelial dysfunction and has been recognized as a risk factor of cardiovascular disease. The present study aimed to investigate the effect of homocysteine (Hcy) on endothelial function in vivo and in vitro, and the underlying signalling pathways. 2. The HHcy animal model was established by intragastric administration with l-methionine in rats. Plasma Hcy and nitric oxide (NO) concentration were measured by fluorescence immunoassay or nitrate reductase method, respectively. Vasorelaxation in response to acetylcholine and sodium nitroprusside were carried out on aortic rings. Human umbilical vein endothelial cells (HUVEC) were treated with indicated concentrations of Hcy in the in vitro experiments. Intracellular NO level and NO concentration in culture medium were assayed. The alterations of possible signalling proteins were detected by western blot analysis. 3. l-methionine administration induced a significant increase in plasma Hcy and decrease in plasma NO. Endothelium-dependent relaxation of aortic rings in response to acetylcholine was impaired in l-methionine-administrated rats. The in vitro study showed that Hcy reduced both intracellular and culture medium NO levels. Furthermore, Hcy decreased phosphorylation of endothelial nitric oxide synthase (eNOS) at serine-1177 and phosphorylation of Akt at serine-473. Hcy-induced dephosphorylation of eNOS at Ser-1177 was partially reversed by insulin (Akt activator) and GF109203X (PKC inhibitor). Furthermore, Hcy reduced vascular endothelial growth factor (VEGF) expression in a dose-dependent manner. 4. In conclusion, Hcy impaired endothelial function through compromised VEGF/Akt/endothelial nitric oxide synthase signalling. These findings will be beneficial for further understanding the role of Hcy in cardiovascular disease.

[Construction of an acellular porcine aortic valve].

OBJECTIVE: To prepare a porcine aortic valve (PAV) free of the cellular components. METHODS: The cellular components of porcine PAV were completely removed using trypsin and Triton X-100, and the acellular PAV was examined microscopically with HE staining with its physical and chemical properties assessed. Transmission electron microscopy was used to observe the integrity of the collagen and elastin and the DNA contents in the PAV was detected to confirm the total removal of the cellular components. With the fresh PAV as the control, small pieces of the acellular PAV were implanted into the subcutaneous tissues of 4 rabbits, and 4 weeks after the implantation, the implants were harvested for microscopic observation. RESULTS: The cellular components were effectively removed from the cusps and roots of the PAV by trypsin and TritonX-100, with marked soluble protein loss [(0.24-/+0.04)% vs (0.48-/+0.12)%] and significantly increased water content [(92.2-/+1.5)% vs (89.2-/+1.6)%]. The acellular PAV still maintained good fibrous scaffold structure and the shrinkage temperature and tension at fracture underwent no significantly changes [(67.9-/+1.0) degrees celsius; vs (68.8-/+0.8) degrees celsius; and (489.3-/+19.0) g/mm2 vs (540.7-/+19.5) g/mm2, respectively]. The PAVs implanted in rabbits showed only mild tissue reaction with a few infiltrating neutrophils, lymphocytes and plasmocytes observed 4 weeks later. The accelular PAV caused obviously milder inflammatory reactions than fresh PAV. CONCLUSIONS: The acellular PAV prepared by treatment with trypsin and Triton X-100 retains good fibrous scaffold structure and mechanical strength with low antigenicity.

[Study on the influence of the configuration of porcine aortic root on the stentless valve design].

OBJECTIVE: To provide the reference for the stentless aortic valve design with the study of the inner configuration of porcine aortic root. METHODS: The orifice areas of porcine aortic root at 4 levels (OA1 to 4), the average area of leaflets (Sa), the area analogue of leaflets (AA, AA = 1/2PH), the average area analogue of leaflets (AAa), the value PH of the left, right, non coronary leaflets (PHl, PHr, PHn) and the sums of PHs of the left and non-coronary leaflets (PHln) in the fresh and glutaraldehyde and epichlorohydrin-treated porcine aortic valves (20 respectively) were measured and calculated. The linear correlation and regression analysis by SPSS 12.0 was used to analyze the correlation between Sa and AAa, OA and Sa, OA and AAa, PHl, PHr and PHn, PHln and PHr in both groups. RESULTS: The coefficient correlation between Sa and AAa in fresh and treated groups were 0.886 and 0.872 respectively (P < 0.05). The coefficient correlation between OA1 to 4 and AAa were 0.810, 0.851, 0.900, and 0.815 respectively in fresh group (P < 0.05), and were 0.852, 0.888, 0.836, and 0.817 respectively in treated group (P < 0.05). This showed that the degree of correlation between the average area analogue of leaflets and the average area of leaflets, the orifice areas of aortic root were relatively large. Additionally, the equation of linear regression existed between PHln and PHr in treated group as follows: PHr = -1.665 + 0.688 PHln (r = 0.907, P < 0.05), thereby PHr could be predicted by PHln. CONCLUSION: The value of PH of leaflets could represent the spatial configuration of the aortic root, which provided a referred index for the stentless bioprostheses design.

Ginsenoside Rb1 preconditioning protects against myocardial infarction after regional ischemia and reperfusion by activation of phosphatidylinositol-3-kinase signal transduction.

BACKGROUND: Ginsenoside Rb1, a major bioactive component of Panax ginseng, bears various beneficial effects on the cardiovascular system. This study investigated whether ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia-reperfusion injury and its potential mechanism. METHODS: Rats subjected to 45 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: sham-operated, ischemia-reperfusion (I/R), ginsenoside Rb1+I/R, wortmannin(a specific PI3K inhibitor)+I/R, wortmannin drug vehicle (dimethyl sulfoxide, DMSO), wortmannin+sham, ginsenoside Rb1+ wortmannin +I/R. Infarct size was assessed by triphenyltetrazolium chloride staining. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and troponin T levels were also measured. Akt phosphorylation expression was assessed by immunoblotting. RESULTS: Ginsenoside Rb1 preconditioning reduced infarct size compared with that in the I/R group: 30 +/- 2.6% versus 51 +/- 2.7% (p < 0.01). Ginsenoside Rb1 preconditioning also markedly reduced the plasma CK, CK-MB, LDH and troponin T levels in blood. Akt phosphorylation expression increased after ginsenoside Rb1 preconditioning. These effects of ginsenoside Rb1 preconditioning were significantly inhibited by wortmannin. CONCLUSION: This is the first study to demonstrate that ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia and reperfusion injury, partly by mediating the activation of the PI3K pathway and phosphorylation of Akt.

[Effect of Tongxinluo superfine on experimental anginal model (contraction of collaterals) in rat with endothelial dysfunction].

OBJECTIVE: To study the effect of Tongxinluo superfine (TXL) on experimental anginal model induced by Arginine Vasopressin in rats with endothelial dysfunction. METHOD: First, the endothelial dysfunction rat model was made by methionine-induced hyperhomocysteinemia (HHcy). The thoracic aorta were excised, and acetylcholine (Ach)-induced endothelium dependent relaxation and sodium nitroprusside (SNP) induced endothelium-independent relaxation were measured. Total plasma homocysteine (Hcy) concentrations were measured with automated fluorescence polarization immunoassay (FPIA). Enzyme-linked immunosorbent assay (ELISA) was used to detect plasma von Willebrand factor (vWF) level. Plasma nitric oxide (NO) contents were assayed by method of nitrate reductase. Then, the rat model of collaterals contraction (model group) was established by AVP intravenous injection in rats with endothelial dysfunction and the S wave change (DeltaS) and T wave depression in Lead II ECG were used as the index of angina severity. The nitric oxide (NO) contents in serum and the expression of myocardium eNOS mRNA were measured. RESULT: Ach (0. 1-1000 nmol L(-1))-induced endothelium dependent relaxation (EDR) of aortic rings was significantly decreased in HHcy group. The endothelium-independent relaxation induced by SNP (0.001-10 micromol L(-1)) was not significantly different between the two groups. Plasma homocysteine concentrations and vWF levels in rats treated with methionine were higher than those of control group, while NO contents were significantly decreased in HHcy group compared with control. The results showed that L-methionine intake induced hyperhomocysteinemia in rats. Impaired EDR, increased vWF and decreased NO suggested the exist of endothelial dysfunction. DeltaS of model group increased from 1 min to 5 min and T wave of model group depressed at 2 min compared with that of control after the administration of vasopressin (0.5 U kg(-1)). The intragastric administration of TXL inhibited vasopressin-induced S wave change at 4 min and 5 min and T wave depression from 30 s to 3 min after AVP injection. The NO contents in serum and the expression of myocardium eNOS mRNA of TXL group were increased compared with model group. CONCLUSION: Experimental angina induced by AVP injection is more severe in rats with endothelial dysfunction. Tongxinluo Superfine can protect against collaterals contraction in rats maybe by increasing the NO contents in serum and the expression of myocardium eNOS mRNA.

[Effects of modified acellularization process on porcine endogenous retroviruses in porcine aorta valves].

OBJECTIVE: to study the effect of modified acellularization process on porcine endogenous retrovirus (PERV) in porcine aorta valves (PAVs). METHODS: Twenty aortic valves of pig were put into 0.1% trypsin solution, hypotonic and hypertonic TritonX-100, DNAse solution, RNAse solution, and Hanks solution in succession so as to remove the cells. The specimens of PAV were to undergo gross observation and microscopy before and after the acellularization procedure. Fracture test was made. Primers specific for the conservative gag gene of PERV were designed PCR and RT-PCR were used to detect the expression of gag. In addition, 20 samples of native PAV were collected. Peripheral mononuclear cells (PBMCs). Were isolated from 20 samples of porcine peripheral blood. Ten dogs underwent acellularized PAV replacement; 3 months later, samples of the dogs' peripheral blood were collected. Porcine kidney cells of the line PK15 were used as positive controls. RESULTS: Microscopy showed that all the cells were removed from the acellularized PAVs. Histological analysis showed that the major structural components were maintained. There was no significant difference in fracture strength between the native and acellularized PAVs (P > 0.05). PCR and RT-PCR showed a PERV 219 bp DNA fragment, 90%-95% homologous with the published PERV gene, in the genomic DNA of all native PAVs, pig PBMCs, and PK15 cells, but not in the acellularized PAVs and dog PBMCs. CONCLUSION: PERV exists in all native PAVs. The modified acellularization process succeeds in removing all the cell component and PERV in the PAVs, thus preventing cross-species transmission of PERV.

Hyperhomocysteinemia and atherosclerosis.

Arteriosclerosis and its complications, such as heart attack and stroke, are the major causes of death in developed countries. It was believed that age, hyperlipidemia, hypertension, diabetes and smoking are common risk factors for cardiovascular disease. In addition, overwhelming clinical and epidemiological studies have identified homocysteine (Hcy) as a significant and independent risk factor for cardiovascular disease. In healthy individuals, plasma Hcy is between 5 and 10 micromol/L. One cause of severe hypehomocys- teinemia (HHcy) is the deficiency of cystathionine beta-synthase (CBS), which converts Hcy to cystathionine. CBS homozygous deficiency results in severe HHcy with Hcy levels up to 100 to 500 micromol/L. Patients with severe HHcy usually present with neurological abnormalities, premature arteriosclerosis. It has been reported that lowering plasma Hcy improved endothelial dysfunction and reduced incidence of major adverse events after percutaneous coronary intervention. The mechanisms by which Hcy induces atherosclerosis are largely unknown. Several biological mechanisms have been proposed to explain cardiovascular pathological changes associated with HHcy. These include: (1) endothelial cell damage and impaired endothelial function; (2) dysregulation of cholesterol and triglyceride biosynthesis; (3) stimulation of vascular smooth muscle cell proliferation; (4) thrombosis activation and (5) activation of monocytes. Four major biochemical mechanisms have been proposed to explain the vascular pathology of Hcy. These include: (1) autooxidation through the production of reactive oxygen species; (2) hypomethylation by forming SAH, a potent inhibitor of biological transmethylations; (3) nitrosylation by binding to nitric oxide or (4) protein homocysteinylation by incorporating into protein. In summary, our studies, as well as data from other laboratories support the concept that Hcy is causally linked to atherosclerosis, and is not merely associated with the disease. Although folic acid, vitamin B12 and B6 can lower plasma Hcy levels, the long-term effects on cardiovascular disease risk are still unknown and judgments about therapeutic benefits await the findings of ongoing clinical trials.

[Construction of a tissue-engineered valve with decellular porcine aortic valve scaffold in the abdominal aorta of canine].

OBJECTIVE: To explore an experimental method for construction of tissue-engineered heart valve (TEHV) in canine abdominal aorta. METHODS: The decellular porcine aortic valve (PAV) leaflets seeded with canine vessel interstitial cells and endothelial cells (ECs) were implanted into 6 canine abdominal aortas. Valve specimens were obtained respectively at the end of 4, 6, 8 and 10 weeks after implantation were studied for morphology, histology and immunohistochemistry. RESULTS: (1) After 4 weeks implantation, multiple layers of cells grew into peripheral portion of valve scaffold, while new extracellular matrix appeared, and original scaffold tissue was partially absorbed. (2) At the end of 10th week after implantation, the decellular PAV scaffold disappeared completely and was substituted by recipient cells and new extracellular matrix. The interstitial cells in matrix was mainly consisted of fibroblasts and myofibroblast. The matrix was mainly composed by type I, III collagen, some elastic fibers with neutral and acid mucopolysaccharide. (3) Surface of valve leaflets were covered with endothelial cells. CONCLUSIONS: (1) TEHV is primarily constructed with recellularized PAV after implantation into canine abdominal aorta for 10 weeks. (2) Heterotopic implantation into the abdominal aorta is an alternative experimental procedure to study the TEHV.

[Construction and evaluation of the property of decellular porcine aortic valve].

OBJECTIVE: To construct decellular porcine aortic valve (PAV) and to observe the existence of porcine endogenous retrovirus (PERV) and valve scaffold structure before and after implantation. METHODS: (1) Porcine aortic valve was obtained. The cellular components of PAV were completely removed by using detergent and nucleotidase solution combined with alteration of osmosis. (2) The decellular underwent HE staining and light microscopy and detection of its physical and chemical properties. (3) 20 pieces of decellular PAV were implanted into dogs. On e month later blood samples of the dogs were collected. PCR and RT-PCR were used to detect the PERV expression in 20 samples of pig's peripheral blood, 20 fresh PAVs, cultured pig kidney cells of the PK15 line (as positive control), decellular PAVs implanted into the dogs, and 10 samples of dogs' peripheral blood. (4) Small pieces of decellular PAVs were implanted into the subcutaneous tissues of 6 rabbits at the back, 6 pieces for one rabbit, and then extracted by the ends of the 4th, 6th, 8th and 10th week respectively after implantation to undergo HE staining and light microscopy. RESULTS: (1) Almost all cellular components in the PAVs had been removed after decellularization; the soluble protein contents lost markedly [(0.238 +/- 0.038)% vs. (0.484 +/- 0.116)%]; the water content of the decellular tissues increased significantly [(92.16 +/- 1.48)% vs. (89.2 +/- 1.55)%]; however, the decellular PAVs still maintained their excellent fibrous scaffold structure, and their shrinkage temperature and tension at fracture were not significantly changed [(72.0 +/- 0.7) degrees C vs. (71.2 +/- 0.8) degrees C, and (448.7 +/- 18.65)g/mm2 vs. (540.7 +/- 19.46)g/mm2 respectively]. (2) Agarose gel electrophoresis of all fresh PAVs and porcine peripheral blood samples showed a 219 bp band, which was 90% to 97% homologous with PERV-C gene, and the sequence of which is published in Medline. No 219 bp amplified band was found in all decellular PAVs and the peripheral blood samples of the dogs implanted with decellular PAV one month after the implantation. (3) The PAVs implanted in rabbit body showed very slight tissue reaction. Neutrophil, lymphocyte and plasmacyte infiltration were seen 4 weeks after; such inflammatory cell infiltration decreased markedly and the peripheral portions of the decellular PAVs began to be absorbed by the end of the 6th week after implantation. The decellular PAVs were completely absorbed without fibrosis or scar formation in the implantation area by the end of the 10th week. CONCLUSION: (1) The cellular components of PAV can be completely removed, the excellent fibrous scaffold structure and mechanical strength of aorta valve can be maintained, and the antigenicity is very weak. Subcutaneous implantation investigation shows that decellular PAV is an absorbable and degradable biological material. (2) There is PERV-C in PAV that can be removed after decellularization. PERV-C reaction is negative in the peripheral blood samples of the recipients implanted with decellular PAV.