Gene therapy in peripheral artery disease.

In the last decade, studies of the biological mechanisms underlying angiogenesis, i.e. the development of a new vasculature from pre-existing blood vessels, have suggested a new approach to peripheral obstructive artery disease based on the treatment of ischemic tissues with angiogenic growth factors. As demonstrated by experimental studies in animal models, a therapeutic effect can be reached as the newly formed vascular network, functioning as a biologic by-pass, restores a normal blood supply to the ischemic territories. New techniques of gene therapy proved effective in reaching sustained concentrations of angiogenic factors in the target tissues. This review concerns the pre-clinical background and the results of the early clinical trials of angiogenic gene therapy, which have shown the safety and feasibility of this new approach.

PGE(1) short term therapy in critical lower limb ischemia.

AIM: Our study aims to evaluate the efficiency of short-term therapy with alprostadil (a PGE molecular derivative) on patients affected by critical ischemia of the lower limbs and unsuitable for surgical revascularization. The study was carried out on two groups of patients treated with the traditional long-term or a short-term protocol respectively. METHODS: The parameters evaluated and statistically compared to existing studies were, the side effects, subjective pain measured on an analogic scale, objective pain calculated according to analgesic intake, and change in trophic lesions. RESULTS: Our results revealed some differences between the two groups. The manifestation of side effects led to treatment suspension in 8% of long-term therapy cases only. Subjective pain was reduced or disappeared in 83.83% of cases (p<0.001) and there was no statistically significant difference between the two groups. The course of analgesic intake was again similar in both groups. Trophic lesions improved or completely healed in 64.7% (p<0.005) and although a greater tendency towards complete healing was evident in the short-term patients, it was not statistically significant. There was no significant difference between the two groups in the ankle/arm pressure index, but a significant improvement has been observed in 30.88% of cases. The results we obtained from both groups are similar and confirm the valid therapeutic use of alprostadil in patients with peripheral arterial occlusive disease (PAOD). CONCLUSION: Our study reveals the presence of intrinsic advantages to the physician with the short-term schedule, through its higher tolerability, improved and more frequent patient and therapy controls and shorter hospitalization.

Remifentanil conscious sedation during regional anaesthesia for carotid endarterectomy: rationale and safety.

OBJECTIVES: to prospectively evaluate the safety and efficacy of remifentanil during regional anaesthesia for carotid endarterectomy. METHODS: twenty-eight consecutive patients underwent carotid endarterectomy with combined superficial and deep cervical plexus block supplemented with continuous intravenous 0.04 microg.kg(-1).min(-1)remifentanil infusion. Depth of sedation was monitored using the Observer's Assessment of Alertness/Sedation Scale (OAA/S). The degree of pain, discomfort and anxiety was self-assessed by the patients using a horizontal visual analogue scale. RESULTS: all patients experienced adequate comfort and analgesia. No local anaesthetic supplementation was necessary. No patient had a OAA/S score lower than 4 (with 5=awake/alert to 1=asleep). Respiratory depression did not occur. Selective shunting was required in four cases. No patient was converted to general anaesthesia. There were no permanent neurological deficits, cardiopulmonary complications or deaths. CONCLUSION: remifentanil as a supplement to regional anaesthesia for carotid endarterectomy, provides comfort and analgesia without hampering mental status evaluation.

Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses tumorigenesis of experimental intracranial human malignant glioma.

Adenoviral-mediated gene transfer for the treatment of experimental intrinsic malignant brain neoplasms holds promise. The role, however, of intracellular, adenoviral-mediated p53 expression to inhibit growth of experimental human intracranial malignant gliomas remains largely unexplored. Using the AdCMV.p53 vector we measured the in vitro expression of p53 and the resultant effect upon U251 human malignant glioma cellular proliferation. We further measured the survival of nude mice after intracranial injection of the infected vs. control U251 cells. The growth of the infected U251 cells was inhibited when compared to both the uninfected cells and cells infected with the control vector (AdCMV.Null). Agarose gel electrophoresis confirmed the AdCMV.p53-dependent cellular apoptosis. Nude mice having intracranial injections of the U251 cells infected with the control (AdCMV.Null) vector showed diminished survival. In contrast, mice having intracranial injections of the cells infected with the AdCMV.p53 vector showed 100% survivorship measured 100 days after treatment. Gene therapy via the AdCMV.p53 viral vector holds promise for the clinical treatment of human malignant gliomas.

Adenovirus-mediated wild-type p53 overexpression inhibits endothelial cell differentiation in vitro and angiogenesis in vivo.

Gene therapy with the tumor suppressor gene p53 induces cancer cell apoptosis in vitro and in vivo and inhibits tumor growth in nude mice. We hypothesized that, in addition to cancer cell apoptosis, a replication-deficient adenovirus vector which carries the cDNA for human wild-type p53 (AdCMV.p53) may also modulate endothelial cell function and inhibit angiogenesis. Human umbilical vein endothelial cells (HUVEC) were infected at different multiplicities of infection (MOI) with either AdCMV.p53, the control vector AdCMV.null or were not infected. Western blot analysis showed p53 overexpression up to 7 days after infection with AdCMV.p53. HUVEC proliferation was either not affected (20 and 50 MOI) or inhibited to comparable levels (100 MOI; P < 0.05) in AdCMV.p53- and AdCMV.null-infected versus uninfected cells. HUVEC differentiation into capillary-like structures on reconstituted basement membrane proteins (Matrigel) was assessed 48 h after infection (100 MOI). After 18 h on Matrigel the capillary-like network formed by AdCMV.p53-infected HUVEC was less extensive than that formed by both AdCMV.null-infected and uninfected control cells (P < 0.05 versus either control). In contrast, conditioned medium from AdCMV.p53-infected HUVEC did not modulate endothelial cell differentiation on Matrigel. The effect of AdCMV.p53 on angiogenesis in vivo was assessed by injecting this vector subcutaneously in mice; 3 days later Matrigel containing basic fibroblast growth factor (bFGF) was injected at the same site. In other experiments AdCMV.p53 was injected simultaneously with an Ad vector coding for vascular endothelial growth factor (AdCMV.VEGF165) into the rat perirenal fat tissue. AdCMV.p53 significantly inhibited neovascularization induced by bFGF within the Matrigel plugs (P < 0.05) or by AdCMV.VEGF165 in the fat tissue (P < 0.05). Thus, the anti-angiogenic effect of Ad-mediated wild-type p53 overexpression may contribute to the ability of this viral vector to inhibit tumor growth.

p53 Induces myocyte apoptosis via the activation of the renin-angiotensin system.

The mechanism by which p53 activates apoptosis in various cell systems is unknown. In the absence of an external death stimulus, p53 and p53-dependent genes, bcl-2 and bax, cannot trigger apoptosis. However, p53 may enhance not only transcription of bax and repress bcl-2, but also may upregulate the local renin-angiotensin system, inducing the formation and secretion of angiotensin II from the cells. To test this hypothesis, adult rat ventricular myocytes were infected with AdCMV.p53, which resulted in downregulation of Bcl-2, upregulation of Bax, and death of 34% of the cells. Gel retardation assays demonstrated p53 binding in the promoters of angiotensinogen and angiotensin II AT1 receptor subtype. Angiotensinogen and AT1 mRNAs increased in AdCMV.p53 cells and this phenomenon was associated with a 14-fold increase in the secretion of angiotensin II. The AT1 receptor blocker losartan and angiotensin II antibody prevented p53-induced apoptosis. Thus, p53 enhances the myocyte renin-angiotensin-system and decreases the Bcl-2/Bax ratio in the cells, triggering apoptosis. The identification of this new pathway in p53-mediated apoptosis may be critical in the alterations of myocardial function in the pathologic heart.

p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.

p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

The tumor suppressive effect of p53 is believed to be rooted in its two primary functions: the implementation of cellular growth arrest and the execution of apoptotic cell death. While p53-regulated expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) appears to be central for the implementation of G1 arrest, the participation of p21(Waf1/Cip1) in p53-triggered cell death remains controversial. In the present study, overexpression of p53 in human melanoma SK-MEL-110 cells through use of an adenoviral expression vector (AdCMV.p53) was found to result in apoptosis, while similar infection of primary vascular smooth muscle cells (VSMC) instead resulted in a moderate inhibition of growth. Expression of p21(Waf1/Cip1) was strongly elevated in VSMC, but showed little change in SK-MEL-110 cells, although expression of another p53-regulated gene (GADD45) was comparable in both AdCMV.p53-infected cell types. Evidence that p21(Waf1/Cip1) expression may be required for surviving p53-induced cell death was further supported by the finding that p53 overexpression was highly toxic for p21-deficient mouse embryonal fibroblasts (p21-/- MEFs). In both SK-MEL-110 and p21-/- MEFs, adenovirus-driven ectopic expression of p21(Waf1/Cip1) resulted in a substantial protection against p53-induced apoptosis, indicating that p21(Waf1/Cip1) rescued cells from a path of programmed cell death to one of enhanced survival.

Anti-tumor gene therapy.

Gene therapy as an anti-tumor strategy is becoming a powerful tool for cytokine delivery to inhibit the growth of many tumors. Several delivery systems are being utilized and designed for the expression of specific genes to achieve a therapeutic result. Liposomes, retroviral vectors, and adenoviral vectors have all been used and eventual clinical application may depend on the type of tumor, the location, the specific gene carried, and the patient's health status. Novel expression vectors may eventually achieve tissue-specific targeting and low immuno-reactivity. Inactivation of mutated oncogenes, such as ras, or re-expression of inactive suppressor genes, such as p53 have been used as strategies for anti-tumor therapy. Additionally, exogenious genes, such as viral thymidine kinase that metabolize chemotherapeutic agents to achieve local cytotoxicity have also been employed. Neuro-endocrine tumors are targets of these gentic strategies since they are often difficult to treat by conventional methods because of their location (brain tumors) or because they have spread from the primary tumor (melanoma). Further advances in the design of these vectors may achieve safe targeting of a variety of malignant tumors.

Safety and efficacy of in vivo gene transfer into the porcine heart with replication-deficient, recombinant adenovirus vectors.

Gene transfer with replication-deficient recombinant adenovirus (Ad) vectors may provide a novel approach to the treatment of some cardiac disorders. The relative efficiency of intramyocardial vs intracoronary Ad vector injection in transducing myocardial cells remains to be determined. Further, Ad vectors are associated with localized inflammation, and this could be associated with clinically significant side-effects. Female minipigs underwent open chest surgery and the Ad vector AdCMV.NLS beta-gal was injected into the circumflex coronary artery (IC; 2 x 10(10) p.f.u.; n = 5) or the posterobasal wall of the left ventricle (i.m.; 5 x 10(9) p.f.u., n = 4; 2 x 10(10) p.f.u., n = 18). The minipigs were killed after 2-31 days and the hearts examined for evidence of beta-galactosidase activity. Minipigs underwent epicardial echocardiography immediately before, within 15 min following the i.m. injection of AdCMV.NLS beta-gal and again at the time of death. Blood samples for white blood cell count, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine and electrolytes were obtained before i.m. and i.c. injection of the Ad vector and before death. Intramuscular injection of the Ad vector was more efficient than i.c. infusion in infecting cells in a localized area of the heart. Myocardial beta-gal activity peaked at 3-6 days after i.m. injection and returned to its control value within 1 month. Although inflammatory cells were present at the injection site, echocardiograms did not show any evidence of either segmental or global left ventricular dysfunction. No minipigs died and all blood tests remained within normal limits following either i.m. or i.c. exposure to the Ad vector. In summary, direct i.m. administration of replication-deficient, recombinant Ad vectors provides a safe and effective approach for short-term gene transfer into the heart of large mammals.

Adenovirus-mediated gene transfer of wild-type p53 results in melanoma cell apoptosis in vitro and in vivo.

Gene transfer techniques may provide efficient treatment for a variety of malignant neoplasms. A replication-deficient adenovirus (Ad) vector which carries the cDNA for wild-type p53 (AdCMV.p53) was tested for its in vitro and in vivo effects on the growth of murine melanoma cell line B16-G3.26 and human melanoma cell line SK-MEL-24. The growth of B16-G3.26 cells infected with AdCMV.p53 was inhibited when compared to the uninfected cells or cells infected with the control vector AdCMV.NLS beta gal. Similarly, the growth of SK-MEL-24 cells infected with AdCMV.p53 was also below that of AdCMV.NLS beta gal-infected and uninfected controls. DNA laddering using agarose gel electrophoresis and in situ labeling of DNA fragmentation (TUNEL) showed that AdCMV.p53-infected murine and human melanoma cells underwent apoptosis. Nude mice injected s.c. either with B16-G3.26 cells or with SK-MEL-24 cells developed localized tumors. These tumors were subsequently infiltrated with either AdCMV.p53, AdCMV.NLS beta gal or saline alone. One week after infection, B16-G3.26 tumors exposed to AdCMV.p53 were 2.5 times smaller than control tumors and exhibited DNA fragmentation. A similar growth-inhibitory effect of AdCMV.p53 was observed with SK-MEL-24 tumors. Thus, Ad-mediated wild-type p53 overexpression resulted in melanoma cell apoptosis and inhibition of melanoma growth in vitro and in vivo. These gene therapy approaches may be useful in targeting rapidly growing, malignant melanomas in a clinical setting.

Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic tumor cells.

The use of replication-deficient adenoviral vectors in gene therapy may become a powerful method to achieve efficient but safe transfer of anti-tumor agents. Introduction of the wild-type p53 gene into tumor cells has, in general, been associated with growth suppression. In this study, infection of androgen-independent human prostate Tsu-pr1 cells lacking functional p53 alleles resulted in high levels of p53 protein within 10-15 h. Cells infected with AdCMV.p53 detached from the substratum, condensed, and exhibited fragmentation of nuclear DNA into nucleosomal units consistent with the process of apoptosis. These effects were evident within 24 h after infection, and the majority of cells had undergone apoptosis by 48 h, whereas cells infected with AdCMV.NLS beta Gal continued to proliferate. Uninfected or AdCMV.NLS beta Gal-infected Tsu-pr1 cells formed tumors in nude mice within 3 weeks after implantation, whereas AdCMV.p53-infected cells failed to form tumors during this period. Therefore, adenoviral-mediated antitumor therapy using the p53 gene is an efficient method to inhibit prostate tumor growth, and agents that target the cellular programmed cell death pathway may be useful in clinical applications.

Comparison between alpha-adrenergic- and K-opioidergic-mediated inositol (1,4,5)P3/inositol (1,3,4,5) P4 formation in adult cultured rat ventricular cardiomyocytes.

In adult cultured rat ventricular cardiac myocytes, both the alpha-adrenergic agonist phenylephrine and the selective kappa opioid receptor ligand U-50, 488H affected phosphoinositide turnover. Phenylephrine, over a time course of 10 min, caused a transient increase in Ins(1,4,5)P3 which peaked at 1 min and had returned to control at 2 min. In addition, phenylephrine produced a progressive and sustained increase in the formation of Ins (1,3,4,5)P4 which achieved a plateau after 5 min of exposure to the agonist. U-50,488H induced an increase in Ins(1,4,5)P3 which peaked at 1 min at a level significantly higher than that due to phenylephrine and was still elevated after 10 min exposure to the kappa opioid receptor agonist. In addition, U-50,488H caused a sustained increase in Ins(1,3,4,5)P4 which was comparable to that due to phenylephrine. The stimulatory effects produced by phenylephrine and U-50,488H were receptor-mediated events, since they were fully antagonized by their respective antagonists, phentolamine or Mr-1452.