A 3D-Printed Assemblable Bespoke Scaffold as a Versatile Microcryogel Carrier for Site-Specific Regenerative Medicine.

Advances in additive manufacturing have led to diverse patient-specific implant designs utilizing computed tomography, but this requires intensive work and financial implications. Here, Digital Light Processing is used to fabricate a hive-structured assemblable bespoke scaffold (HIVE). HIVE can be manually assembled in any shape/size with ease, so a surgeon can create a scaffold that will best fit a defect before implantation. Simultaneously, it can have site-specific treatments by working as a carrier filled with microcryogels (MC) incorporating different biological factors in different pockets of HIVE. After characterization, possible site-specific applications are investigated by utilizing HIVE as a versatile carrier with incorporated treatments such as growth factors (GF), bioceramic, or cells. HIVE as a GF-carrier shows a controlled release of bone morphogenetic protein/vascular endothelial growth factor (BMP/VEGF) and induced osteogenesis/angiogenesis from human mesenchymal stem cells (hMSC)/human umbilical vein endothelial cells (HUVECs). Furthermore, as a bioceramic-carrier, HIVE demonstrates enhanced mineralization and osteogenesis, and as a HUVEC carrier, it upregulates both osteogenic and angiogenic gene expression of hMSCs. HIVE with different combinations of MCs yields a distinct local effect and successful cell migration is confirmed within assembled HIVEs. Finally, an in vivo rat subcutaneous implantation demonstrates site-specific osteogenesis and angiogenesis.

Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc.

PURPOSE: Hyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease. METHODS: Gene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1ß, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression. RESULTS: Gene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor-donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls. CONCLUSION: Although HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target. These slides can be retrieved under Electronic Supplementary Material.

TRPC6 in simulated microgravity of intervertebral disc cells.

PURPOSE: Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. However, the underlying molecular mechanisms are not completely understood. Transient receptor potential canonical (TRPC) channels are implicated in mechanosensing of several tissues, but are poorly explored in IVDs. METHODS: Primary human IVD cells from surgical biopsies composed of both annulus fibrosus and nucleus pulposus (passage 1-2) were exposed to simulated microgravity and to the TRPC channel inhibitor SKF-96365 (SKF) for up to 5 days. Proliferative capacity, cell cycle distribution, senescence and TRPC channel expression were analyzed. RESULTS: Both simulated microgravity and TRPC channel antagonism reduced the proliferative capacity of IVD cells and induced senescence. While significant changes in cell cycle distributions (reduction in G1 and accumulation in G2/M) were observed upon SKF treatment, the effect was small upon 3 days of simulated microgravity. Finally, downregulation of TRPC6 was shown under simulated microgravity. CONCLUSIONS: Simulated microgravity and TRPC channel inhibition both led to reduced proliferation and increased senescence. Furthermore, simulated microgravity reduced TRPC6 expression. IVD cell senescence and mechanotransduction may hence potentially be regulated by TRPC6 expression. This study thus reveals promising targets for future studies. These slides can be retrieved under Electronic Supplementary Material.

Inflammaging in cervical and lumbar degenerated intervertebral discs: analysis of proinflammatory cytokine and TRP channel expression.

PURPOSE: To investigate and compare the occurrence of inflammatory processes in the sites of disc degeneration in the lumbar and cervical spine by a gene array and subsequent qPCR and to investigate the mechanistic involvement of transient receptor potential channels TRPC6 and TRPV4. METHODS: The gene expression of inflammatory cytokines and TRP channels was measured in human disc samples obtained from patients undergoing discectomy at the cervical (n = 24) or lumbar (n = 27) spine for degenerative disc disease (DDD) and disc herniation (DH) and analyzed for differences with regard to spinal level, IVD degeneration grade, Modic grade, age, sex, disc region and surgical extent. RESULTS: Aside from genes with known implication in DDD and DH, four previously unreported genes from the interferon and TRP families (IFNA1, IFNA8, IFNB1, TRPC6) could be detected. A correlation between gene expression and age (IL-15) and IVD degeneration grade (IFNA1, IL-6, IL-15, TRPC6), but not Modic grade, was identified. Significant differences were detected between cervical and lumbar discs (IL-15), nucleus and annulus (IL-6, TNF-α, TRPC6), single-level and multi-level surgery (IL-6, IL-8) as well as DDD and DH (IL-8), while sex had no effect. Multiple gene-gene pair correlations, either between different cytokines or between cytokines and TRP channels, exist in the disc. CONCLUSION: This study supports the relevance of IL-6 and IL-8 in disc diseases, but furthermore points toward a possible pathological role of IL-15 and type I interferons, as well as a mechanistic role of TRPC6. With limited differences in the inflammatory profile of cervical and lumbar discs, novel anti-inflammatory or TRP-modulatory strategies for the treatment of disc pathologies may be applicable independent of the spinal region.

Epigenetic regulation of tissue factor inducibility in endothelial cell senescence.

Cellular senescence, a programmed state induced by multiple deleterious triggers, is characterised by permanent cell-cycle exit and altered gene expression and cell morphology. In humans it is considered a tumor suppressor mechanism, mediating removal of damaged or mutated cells from the cell-cycle pool, and may also contribute to the ageing process. In this study, we show that senescent human umbilical vein endothelial cells lose their ability to induce tissue factor (TF), a transmembrane protein with important roles in hemostasis and cancer progression, in response to thrombin or - independently of cell-surface receptors - phorbol-12-myristate-13-acetate. This phenomenon could not be explained by senescence-related alterations in the downstream signal transduction cascade or by accelerated TF mRNA degradation. Rather, using chromatin immuno-precipitation we could show that loss of TF gene inducibility during senescence occurs following chromatin remodelling of the TF promoter resulting from hypo-acetylation of histone H3. These findings were reversible after transduction of presenescent cultures with telomerase reverse transcriptase, enabling late-passage cultures to escape senescence. These results extend the involvement of heterochromatic gene silencing in senescence beyond cell cycle-related genes and suggest a novel anti-cancer mechanism of senescence through inhibition of TF inducibility.

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition.

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.

Endothelial nitric oxide synthase gene transfer inhibits human smooth muscle cell migration via inhibition of Rho A.

Smooth muscle cell (SMC) migration contributes to vascular remodeling. Nitric oxide (NO) produced via endothelial NO synthase (eNOS) inhibits SMC migration. This study analyzes signal transduction mechanisms of SMC migration targeted by NO. SMCs were cultured from human saphenous veins, and cell migration was studied using Boyden chambers. PDGF-BB (0.1 to 10 ng/ml) stimulated SMC migration in a concentration-dependent manner, which was inhibited by adenoviral-mediated overexpression of eNOS and by the NO donor diethylentriamine NONOate (DETANO, 10 to 10 mol/L). NO release was enhanced in eNOS-transduced SMCs, and L-NAME blunted the effect of eNOS overexpression on migration. PDGF-BB (10 ng/ml) activated Rho A, which was inhibited by the overexpression of eNOS by DETANO and by 8 bromo-cGMP. The inhibitory effect of DETANO on Rho A activity was prevented by the cGMP-dependant kinase inhibitor. Furthermore, inhibition of Rho A by C3 exoenzyme and inhibition of ROCK by Y-27632 diminished cell migration stimulated by PDGF-BB. Finally, in the cells overexpressing constitutively active ROCK mutant (CAT), DETANO failed to prevent PDGF-BB-induced SMC migration. In conclusion, NO inhibits human SMC migration via blockade of the Rho A pathway.

Inactivity of nitric oxide synthase gene in the atherosclerotic human carotid artery.

OBJECTIVE: Nitric oxide (NO) inhibits thrombus formation, vascular contraction, and smooth muscle cell proliferation. We investigated whether NO release is enhanced after endothelial NO synthase (eNOS) gene transfer in atherosclerotic human carotid artery ex vivo. METHODS AND RESULTS: Western blotting and immunohistochemistry revealed that transduction enhanced eNOS expression; however, neither nitrite production nor NO release measured by porphyrinic microsensor was altered. In contrast, transduction enhanced NO production in non-atherosclerotic rat aorta and human internal mammary artery. In transduced carotid artery, calcium-dependent eNOS activity was minimal and did not differ from control conditions. Vascular tetrahydrobiopterin concentrations did not differ between the experimental groups. Treatment of transduced carotid artery with FAD, FMN, NADPH, L-arginine, and either sepiapterin or tetrahydrobiopterin did not alter NO release. Superoxide formation was similar in transduced carotid artery and control. Treatment of transduced carotid artery with superoxide dismutase (SOD), PEG-SOD, PEG-catalase did not affect NO release. CONCLUSIONS: eNOS transduction in atherosclerotic human carotid artery results in high expression without any measurable activity of the recombinant protein. The defect in the atherosclerotic vessels is neither caused by cofactor deficiency nor enhanced NO breakdown. Since angioplasty is performed in atherosclerotic arteries,eNOS gene therapy is unlikely to provide clinical benefit.

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.

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.

Absence of histamine-induced nitric oxide release in the human radial artery: implications for vasospasm of coronary artery bypass vessels.

Radial artery (RA) bypass grafts can develop severe vasospasm. As histamine is known to induce vasospasm, its effect on RA was assessed compared with the classic bypass vessels internal mammary artery (MA) and saphenous vein (SV). The vessels were examined in organ chambers for isometric tension recording. Histamine induced contractions on baseline; the sensitivity was higher in RA and SV than MA. After precontraction with norepinephrine, histamine did not evoke relaxations of RA but induced relaxations of MA and less of SV at lower concentrations; it induced contractions at higher concentrations, reaching similar levels in all three vessels. Indomethacin did not affect the response of MA and RA but potentiated relaxations and reduced contractions of SV. Endothelium removal, N(omega)-nitro-L-arginine methyl ester (L-NAME), or the H2-receptor blocker cimetidine did not affect the response of RA, but inhibited relaxations and enhanced contractions in MA and inhibited relaxations in SV; in the latter, only L-NAME enhanced contractions. Real-time PCR detected much lower expression of endothelial H2-receptor in RA than MA or SV. Western blots revealed similar endothelial nitric oxide (NO) synthase expression in all three vessels. Relaxations to acetylcholine were identical in RA and MA. Thus histamine releases NO by activating the endothelial H2-receptor, the expression of which is much lower in RA than MA or SV. H2-receptor activation also releases prostaglandins in SV, partially antagonizing NO. The lack of histamine-induced NO production represents a possible mechanism of RA vasospasm.

Effects of tacrolimus or sirolimus on proliferation of vascular smooth muscle and endothelial cells.

Local strategies directed against vascular smooth muscle cell (VSMC) proliferation such as drug-eluting stents reduce the occurrence of restenosis. However, these approaches may also inhibit endothelial cell (EC) proliferation and, thus, impair reendothelialization. We compared the effects of tacrolimus on human VSMC and EC proliferation and migration to sirolimus, a compound with similar molecular structure. Thymidine incorporation was determined in growth factor-stimulated VSMC and EC. The drug concentration at which maximal VSMC proliferation was inhibited by 50% (IC50) was about 10-fold higher for tacrolimus (3.8 x 10 M) than for sirolimus (4.1 x 10 M; P = 0.055). It is interesting that the molar IC50 value in EC was around 10-fold higher for tacrolimus (2.3 x 10 M) than for sirolimus (7.1 x 10 M; P < 0.01). The profile of these drugs on VSMC and EC migration was similar to the one found in the proliferation assays. Inhibition of VSMC proliferation by both tacrolimus and sirolimus was associated with upregulation of the cell-cycle inhibitor p27. Thus, tacrolimus is less potent than sirolimus for inhibiting VSMC proliferation or migration. However, tacrolimus exerts markedly less antiproliferative effects on EC compared with sirolimus. In combination with its potent antiinflammatory effects, tacrolimus may represent a promising compound for the use in drug-eluting stents.

Histamine induces tissue factor expression: implications for acute coronary syndromes.

BACKGROUND: Histamine can induce coronary vasospasm, leading to variant angina and acute myocardial infarction. However, the role of histamine in thrombus formation is ill defined. Hence, this study investigates whether histamine induces tissue factor (TF) expression in vascular cells. METHODS AND RESULTS: Histamine (10(-8) to 10(-5) mol/L) induced TF expression in a concentration-dependent manner in human aortic endothelial and vascular smooth muscle cells, whereas TF pathway inhibitor expression remained unaffected. RT-PCR and Northern blotting revealed that histamine stimulated TF mRNA transcription, peaking at 1 hour. Protein expression increased 18-fold (P<0.02) with a maximum at 5 hours, which was paralleled by a 4-fold augmentation in surface activity (P<0.01). These effects were completely prevented by pretreatment with the H1 receptor antagonists mepyramine (P<0.0001), chlorpheniramine, and diphenhydramine but not the H2 receptor antagonist cimetidine (P=NS). Histamine induced a time-dependent, H1 receptor-mediated activation of p38 MAP kinase (p38), p44/42 MAP kinase (ERK), and c-jun terminal NH2 kinase (JNK). Blocking of p38, ERK, or JNK with SB203580 (P<0.0001), PD98059 (P<0.0001), or SP600125 (P<0.0001), respectively, impaired histamine-induced TF expression in a concentration-dependent manner. In contrast, histamine-stimulated TF expression was increased by phosphatidylinositol 3-kinase inhibition with LY294002 or wortmannin, whereas it was not affected by Rho-kinase inhibition with Y-27632 or hydroxyfasudil. CONCLUSIONS: Histamine induces expression of TF, but not TF pathway inhibitor, in vascular cells via activation of the H1, but not H2, receptor. This effect is mediated by the MAP kinases p38, ERK, and JNK. This observation may open novel perspectives in the treatment of variant angina and acute coronary syndromes.

Celecoxib decreases endothelial tissue factor expression through inhibition of c-Jun terminal NH2 kinase phosphorylation.

BACKGROUND: Despite potential antiinflammatory properties, the use of selective cyclooxygenase-2 inhibitors (coxibs) in patients with cardiovascular diseases has been questioned because of a possibly increased thrombotic risk. Tissue factor (TF), a key protein for initiation of coagulation, has been implicated in the pathogenesis of atherosclerosis and thrombosis. Hence, we examined the effect of different coxibs on TF expression. METHODS AND RESULTS: Celecoxib (10(-5) mol/L), but not rofecoxib (10(-7) to 10(-5) mol/L) or the experimental coxib NS-398 (10(-7) to 10(-5) mol/L), decreased tumor necrosis factor-alpha-induced TF expression and activity in human aortic endothelial cells. Celecoxib (10(-5) mol/L) reduced activation of c-jun terminal NH2 kinase (JNK), whereas it did not affect p38 mitogen-activated protein (MAP) kinase or p44/42 MAP kinase; in contrast, JNK activation was not affected by rofecoxib (10(-5) mol/L) or NS-398 (10(-5) mol/L). TF expression was reduced in a concentration-dependent manner by pretreatment with SP600125 (10(-7) to 10(-6) mol/L), a specific inhibitor of JNK, which confirms that JNK regulates tumor necrosis factor-alpha-induced TF expression. CONCLUSIONS: Celecoxib reduced TF expression and activity in human aortic endothelial cells. Because neither rofecoxib nor the experimental coxib NS-398 affected TF expression, this effect occurs independently of COX-2 inhibition; it is rather mediated through inhibition of JNK phosphorylation. These data indicate a distinct heterogeneity within this class of drugs, which may be clinically relevant, especially for patients with atherosclerotic vascular diseases.

Nitric oxide synthase gene transfer inhibits biological features of bypass graft disease in the human saphenous vein.

BACKGROUND: Bypass graft disease is related to proliferation and migration of vascular smooth muscle cells and to platelet activation with thrombus formation. Nitric oxide inhibits these biological responses; it has never been demonstrated, however, whether this occurs in intact human vascular tissue after endothelial nitric oxide synthase gene transfer. METHODS: We examined whether endothelial nitric oxide synthase overexpression inhibits biological features of bypass graft disease in saphenous vein tissue. RESULTS: The nitric oxide donor diethylenetriamineNONOate inhibited proliferation (P <.001) and migration (P <.001) of human saphenous vein vascular smooth muscle cells in response to 20% serum in a concentration-dependent manner. A similar effect on proliferation (P <.05) and migration (P <.05) without any cytotoxicity was observed after adenoviral endothelial nitric oxide synthase transfection. Staining of saphenous vein tissue for placental alkaline phosphatase demonstrated that adenoviral transfection was efficient. Consistent with this observation, endothelial nitric oxide synthase protein expression and nitric oxide release were enhanced in transfected tissue. Further, endothelial nitric oxide synthase overexpression inhibited vascular smooth muscle cell outgrowth from saphenous vein explants over 21 days; 48% +/- 12% of explants exhibited outgrowth after treatment with endothelial nitric oxide synthase adenovirus as compared with 69% +/- 10% in those infected with control adenovirus and 90% +/- 5% in uninfected tissue (P <.05). Similarly, platelet adhesion to human saphenous vein tissue was inhibited by endothelial nitric oxide synthase overexpression; adhesion was reduced in segments infected with endothelial nitric oxide synthase adenovirus (58% +/- 6%) as compared with those infected with control adenovirus (107% +/- 8%) or uninfected saphenous vein (100%; P <.05). CONCLUSIONS: These data demonstrate that endothelial nitric oxide synthase gene transfer inhibits biological features of bypass graft disease in intact human saphenous vein tissue. Therefore, endothelial nitric oxide synthase transfection represents a promising gene transfer approach to prevent venous bypass graft disease.

Endothelial and smooth muscle cell dysfunction in human atherosclerotic radial artery: implications for coronary artery bypass grafting.

The radial artery (RA) is increasingly used as coronary artery bypass graft. In rare cases, however, it is macroscopically atherosclerotic at time of harvest. We examined how the regulation of vascular tone is altered under such circumstances. Macroscopically evident atherosclerotic lesions were observed in 3 of 429 RA (0.7%) isolated within 2 years. Histology revealed severe plaque formation; however, von Willebrand Factor staining showed a morphologically intact endothelium (n = 3). Endothelium-dependent relaxations to acetylcholine (10(-5) M) were reduced in atherosclerotic RA (32 +/- 6%; n = 3) as compared with control (78 +/- 4%; n = 10; P = 0.0001). Receptor-independent contractions to KCl (100 mM) were reduced in atherosclerotic RA (33.19 +/- 5.06 mN; n = 3) as compared with control (108.02 +/- 15.76 mN; n = 9; P < 0.05). Similarly, contractions to thrombin (1 U/ml) were reduced in atherosclerotic RA (3.02 +/- 1.58 mN; n = 3) as compared with control (10.97 +/- 5.12 mN; n = 8). Likewise, receptor-mediated contractions to norepinephrine were reduced in atherosclerotic RA (27.64 +/- 12.48 mN; n = 3) as compared with control (82.74 +/- 11.36 mN; n = 9; P < 0.05). Atherosclerosis is rare in RA of patients with coronary artery disease, but it does occur. Atherosclerotic RA exhibits a dysfunction of both endothelium and vascular smooth muscle. This dysfunction may favor thrombus formation and accelerated atherogenesis. Therefore, atherosclerotic RA should not be used for coronary artery bypass grafting.

Different cell cycle regulation of vascular smooth muscle in genetic hypertension.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster than those from Wistar-Kyoto rats (WKY). Therefore regulation of cell cycle progression was examined in VSMC from both strains. Analysis of G1 progression was performed in VSMC synchronized by serum starvation. Double staining for propidium iodide and bromodeoxyuridine revealed that G1 progression was faster in SHR as compared with WKY. Indeed, 59+/-6% of VSMC from SHR but only 14+/-10% of those from WKY had left G1 phase after 24 hours of mitogenic stimulation. Moreover, 15+/-2% of SHR cells had already completed the cycle at this time point. Western blot analysis demonstrated that the level of cyclin D, cyclin E, and cyclin A was higher in SHR cells progressing through G1 phase, whereas expression of cyclin-dependent kinase 2 as well as the cyclin-dependent kinase inhibitors p21 and p27 were similar in the two groups. Consistent with a higher level of cyclins, the activity of cyclin-dependent kinase 2 was more pronounced in SHR cells. Analysis of G2 progression was performed in VSMC synchronized by treatment with aphidicolin and revealed an additional difference in cell cycle regulation between SHR and WKY. Indeed, the level of cell division cycle kinase 2 was higher in cells from SHR, whereas that of its catalytic partner cyclin B was similar. Consistent with this pattern of expression, the activity of cell division cycle kinase 2 was more pronounced in VSMC from SHR as compared with WKY. Thus, these data demonstrate that the different proliferation of VSMC from SHR and WKY is related to a different progression in G1 phase as the result of the expression of cyclin D, cyclin A, and cyclin E as well as a different progression in G2 phase caused by expression of cell division cycle kinase 2.