Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

The synergistic action of a VEGF-receptor tyrosine-kinase inhibitor and a sensitizing PDGF-receptor blocker depends upon the stage of vascular maturation

OBJECTIVE: To investigate the effects of tyrosine-kinase inhibitors of vascular endothelial growth factor (VECF) and platelet-derived growth factor (PDCF)-receptors on non-malignant tissue and whether they depend upon the stage of vascular maturation. MATERIALS AND METHODS: PTK787/ZK222584 and CGP53716 (VEGF- and PDGF-receptor inhibitor respectively), both alone and combined, were applied on chicken chorioallantoic membrane (CAM). RESULTS: On embryonic day of CAM development (E)8, only immature microvessels, which lack coverage of pericytes, are present: whereas the microvessels on E12 have pericytic coverage. This development was reflected in the expression levels of pericytic markers (alpha-smooth muscle actin, PDGF-receptor beta and desmin), which were found by immunoblotting to progressively increase between E8 and E12. Monotherapy with 2 microg of PTK787/ZK222584 induced significant vasodegeneration on E8, but not on E12. Monotherapy with CGP53716 affected only pericytes. When CGP53716 was applied prior to treatment with 2 microg of PTK787/ZK222584, vasodegeneration occurred also on E12. The combined treatment increased the apoptotic rate. as evidenced by the cDNA levels of caspase-9 and the TUNEL-assay. CONCLUSION: Anti-angiogenic treatment strategies for non-neoplastic disorders should aim to interfere with the maturation stage of the target vessels: monotherapy with VEGF-receptor inhibitor for immature vessels, and combined anti-angiogenic treatment for well developed mature vasculature.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

Angiopoietin-2 deficiency decelerates age-dependent vascular changes in the mouse retina

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.

Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.