Two-Stage Gene Therapy (VEGF, HGF and ANG1 Plasmids) as Adjunctive Therapy in the Treatment of Critical Lower Limb Ischemia in Diabetic Foot Syndrome.

One of the most serious problems in people with diabetes is diabetic foot syndrome. Due to the peripheral location of atherosclerotic lesions in the arterial system of the lower extremities, endovascular treatment plays a dominant role. However, carrying out these procedures is not always possible and does not always bring the expected results. Gene therapy, which stimulates angiogenesis, improves not only the inflow from the proximal limb but also the blood redistribution in individual angiosomes. Due to the encouraging results of sequential treatment consisting of intramuscular injections of VEGF/HGF bicistronic plasmids followed by a month of ANG1 plasmids, we decided to use the described method for the treatment of critical ischemia of the lower limbs in the course of diabetes and, more specifically, in diabetic foot syndrome. Twenty-four patients meeting the inclusion criteria were enrolled in the study. They were randomly divided into two equal groups. The first group of patients was subjected to gene therapy, where the patients received intramuscular injections of pIRES/VEGF165/HGF plasmids and 1 month of ANG-1 plasmids. The remaining patients constituted the control group. Gene therapy was well tolerated by most patients. The wounds healed significantly better in Group 1. The minimal value of ABI increased significantly in Group 1 from 0.44 ± 0.14 (± standard deviation) to 0.47 ± 0.12 (with p = 0.028) at the end of the study. There were no significant differences in the control group. In the gene treatment group, PtcO2 increased significantly (from 28.71 ± 10.89 mmHg to 33.9 ± 6.33 mmHg with p = 0.001), while in Group 2, no statistically significant changes were found. The observed resting pain decreased significantly in both groups (Group 1 decreased from 6.80 ± 1.48 to 2.10 ± 1.10; p < 0.001; the control group decreased from 7.44 ± 1.42 to 3.78 ± 1.64 with p < 0.001). In our study, we evaluated the effectiveness of gene therapy with the growth factors described above in patients with CLI in the course of complicated DM. The therapy was shown to be effective with minimal side effects. No serious complications were observed.

Preliminary Assessment of Intra-Aneurysm Sac Pressure During Endovascular Aneurysm Repair as an Early Prognostic Factor of Aneurysm Enlargement.

Purpose: Numerous cases of abdominal aortic aneurysm (AAA) enlargement, and even rupture, despite endovascular aneurysm repair (EVAR), have been documented. This has been linked to increased aneurysm sac pressure (ASP). We decided to conduct further research with the aim to identify correlations between ASP during EVAR and subsequent aneurysm enlargement. Patients and Methods: This experimental prospective study included 30 patients undergoing EVAR of infrarenal AAAs. Invasive ASP measurements were done using a thin pressure wire. Aortic pressure (AP) was measured using a catheter placed over the wire. Systolic pressure index (SPI), diastolic pressure index (DPI), mean pressure index (MPI), and pulse pressure index (PPI) were calculated both for ASP and AP. The results of follow-up computed tomography angiography (CTA) at 3 months were compared with baseline CTA findings. Results: During EVAR, a significant reduction was observed for SPI (from 98% to 61%), DPI (from 100% to 87%), MPI (from 99% to 74%), and PPI (from 97% to 34%). There were no significant correlations of pressure indices with an aneurysm diameter, cross-sectional area, velocity, thrombus shape and size, number of patent lumbar arteries, length and diameter of aneurysm neck, diameter of the inferior mesenteric artery, as well as diameter and angle of common iliac arteries. On the other hand, aneurysm neck angulation was significantly inversely correlated with reduced PPI. After combining CTA findings with pressure measurements, we identified a positive correlation between PPI and aneurysm enlargement (ratio of the cross-sectional area at the widest spot at baseline and at 3 months after EVAR). Conclusion: The study showed that ASP can be successfully measured during EVAR and can facilitate the assessment of treatment efficacy. In particular, PPI can serve as a prognostic factor of aneurysm enlargement and can help identify high-risk patients who remain prior monitoring.

Effect of Reconstructive Procedures of the Extracranial Segment of the Carotid Arteries on Damage to the Blood-Brain Barrier.

INTRODUCTION: Endarterectomy and angioplasty of the internal carotid artery are surgical measures for the prevention of ischemic stroke. Perioperative complications are caused by concomitant embolism and reperfusion syndrome leading to damage of the blood-brain barrier. METHODS: The study included 88 patients divided into two groups, depending on the surgical technique used: internal carotid artery endarterectomy (CEA), 66 patients, and percutaneous carotid angioplasty and stenting (CAS), 22 patients. Blood was drawn 24 h before surgery, as well as 8, 24, and 48 h post-surgery. The assessment of damage to the blood-brain barrier was based on the evaluation of the concentration of claudin-1 and occludin, aquaporin-4, the measurements of the activity of metalloproteinase-2 (MMP-2) and -9 (MMP-9), and the assessment of central nervous system damage, measured by changes in the blood S100ß protein concentration. RESULTS: A significant increase in the concentration of the blood-brain barrier damage markers and increased MMP-2 and MMP-9 activity were found in patient blood. The degree of damage to the blood-brain barrier was higher in the CEA group. CONCLUSIONS: The authors' own research has indicated that revascularization of the internal carotid artery may lead to damage to the central nervous system secondary to damage to the blood-brain barrier.

Local intramuscular administration of ANG1 and VEGF genes using plasmid vectors mobilizes CD34+ cells to peripheral tissues and promotes angiogenesis in an animal model.

INTRODUCTION: Patients with peripheral artery disease have poor prognosis despite advances in vascular surgery. Therefore, attempts have been made at using gene and cell therapy to stimulate angiogenesis in the lower limbs in patients with critical lower limb ischemia (CLI). METHODS: The study included 30 rats divided into 3 groups. An intramuscular injection of a therapeutic gene or cells in the right hind limb was administered in each group: angiopoietin-1 (ANG1) plasmid in group 1, ANG1/vascular endothelial growth factor (ANG1/VEGF) bicistronic construct in group 2, and naked plasmid in group 3 (control). After 3 months of follow-up, tissue samples were harvested, and vessels that stained positively for CD34 cells were quantified. RESULTS: The highest CD34+ cell count was noted in the ANG1/VEGF group (98.26 cells), followed by the ANG1 group (80.31) and control group (47.93). The CD34+ cell count was significantly higher in the ANG1/VEGF and ANG1 groups than in the control group. There was no significant difference in the CD34+ cell count between the ANG1/VEGF and ANG1 groups. CONCLUSION: Our study confirmed that therapy with ANG1 plasmid alone or ANG1/VEGF bicistronic construct is safe and effective in a rat model. The therapy resulted in the recruitment of more CD34+ vascular endothelial cells than in the control group receiving naked plasmid.

Double VEGF/HGF Gene Therapy in Critical Limb Ischemia Complicated by Diabetes Mellitus.

Critical leg ischemia (CLI) complicated by diabetes mellitus (DM), which is a very common and dangerous disease, represents the ultimate stage of peripheral arterial disease. Patients are treated with antiplatelet drugs, statins and limb revascularization, but a significant number of patients are not candidate for revascularization. Literature shows that in such cases, gene therapy could be a perfect therapeutic option. The aim of our study was to evaluate efficacy of double vascular endothelial growth factor/hepatocyte growth factor (VEGF/HGF) gene therapy in patients with CLI complicated by DM. We observed that 90 days after administration, serum level of VEGF and ankle-brachial index increased significantly (p < 0.001) and rest pain decreased significantly compared with the control group (p < 0.002). Moreover considerable improvement in vascularization was observed in computed tomography angiography (P = 0.04). Based on the results of this study, we suggest that the therapy with pIRES/VEGF165/HGF bicistronic plasmid administration is a safe and effective method of treatment of patients with both CLI and DM. Graphical abstract.

Treatment of Critical Limb Ischemia by pIRES/VEGF165/HGF Administration.

BACKGROUND: Prognosis of peripheral artery disease (PAD), especially critical limb ischemia (CLI), is very poor despite the development of endovascular therapy and bypass surgery. Many patients result in having leg amputation. We decided to investigate the safety and efficacy of plasmid of internal ribosome entry site/vascular endothelial growth factor (VEGF) 165/hepatocyte growth factor (HGF) gene therapy (GT) in patients suffered from CLI. METHODS: Administration of plasmid of internal ribosome entry site/VEGF165/HGF was performed in 12 limbs of 12 patients with rest pain and ischemic ulcers due to CLI. Plasmid was injected into the muscles of the ischemic limbs. The levels of VEGF in serum and the ankle-brachial index (ABI) were measured before and after treatment. RESULTS: Mean (±SD) plasma levels of VEGF increased nonsignificantly from 258 ± 81 pg/L to 489 ± 96 pg/L (P > 0.05) 2 weeks after therapy, and the ABI improved significantly from 0.27 ± 0.20 to 0.50 ± 0.22 (P < 0.001) 3 months after therapy. Ischemic ulcers healed in 9 limbs. Amputation was performed in 3 patients because of advanced necrosis and wound infection. However, the level of amputations was lowered below knee in these cases. Complications were limited to transient leg edema in 3 patients and fever in 2 patients. CONCLUSIONS: Intramuscular administration of plasmid of internal ribosome entry site/VEGF165/HGF is safe, feasible, and effective for patients with critical leg ischemia.

Role of vascular endothelial growth factor in inducing production of angiopoetin-1 - in vivo study in Fisher rats.

The aim of the study was to investigate how an intramuscular injection of plasmids with genes coding various pro-angiogenic factors: angiopoetin-1 (ANGPT1), vascular endothelial growth factor (VEGF165) and hepatic growth factor (HGF), influences the production of ANGPT1. 40 Healthy Fisher rats received i.m. injections containing plasmids encoding pro-angiogenic genes in thigh muscles. They were divided into four equal groups. The first group received the plANGPT1 plasmid and the second group- the pIRES/ANGPT1/VEGF165 bicistronic plasmid. The pIRES/VEGF165/HGF bicistronic plasmid was administered to the third group and an empty plasmid (control group) to the fourth group. The animals were euthanized after 12 weeks. In each group, the number of vessels stained with the anti-ANGPT1 antibody was assessed under an optical microscope. The anti-ANGPT1 antibodies stained the vessels in all the groups. There were on average 14.1 ±2.3 vessels in the the plANGPT1 group, 32.5 ±10.5 in the pl/RESANGPT1/VEGF group and 30.8 ±13.3 in the plRES/HGV/VEGF group. There were on average 7.3 ±2.3 stained vessels (p < 0.0001) in the control group . The VEGF plays a role in the induction of the production of ANGPT1. The administration of plasmids only encoding ANGPT1 does not induce its production.