Complementary Gene Therapy after Revascularization with the Saphenous Vein in Diabetic Foot Syndrome.

Diabetic foot syndrome (DFS) is one of the most serious macroangiopathic complications of diabetes. The primary treatment option is revascularization, but complementary therapies are still being sought. The study group consisted of 18 patients diagnosed with ischemic ulcerative and necrotic lesions in DFS. Patients underwent revascularization procedures and, due to unsatisfactory healing of the lesions, were randomly allocated to two groups: a group in which bicistronic VEGF165/HGF plasmid was administered and a control group in which saline placebo was administered. Before gene therapy administration and after 7, 30, 90, and 180 days, color duplex ultrasonography (CDU) was performed, the ankle-brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) were measured, and DFS changes were described and documented photographically. In the gene therapy group, four out of eight patients (50%) healed their DFS lesions before 12 weeks. During this time, the ABI increased by an average of 0.25 and TcPO2 by 30.4 mmHg. In the control group, healing of the lesions by week 12 occurred in six out of nine patients (66.67%), and the ABI increased by an average of 0.14 and TcPO2 by 27.1 mmHg. One major amputation occurred in each group. Gene therapy may be an attractive option for complementary treatment in DFS.

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.

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.

Brain-dead and coma patients exhibit different serum metabolic profiles: preliminary investigation of a novel diagnostic approach in neurocritical care.

There is a clear difference between severe brain damage and brain death. However, in clinical practice, the differentiation of these states can be challenging. Currently, there are no laboratory tools that facilitate brain death diagnosis. The aim of our study was to evaluate the utility of serum metabolomic analysis in differentiating coma patients (CP) from individuals with brain death (BD). Serum samples were collected from 23 adult individuals with established diagnosis of brain death and 24 patients in coma with Glasgow Coma Scale 3 or 4, with no other clinical symptoms of brain death for at least 7 days after sample collection. Serum metabolomic profiles were investigated using proton nuclear magnetic resonance (NMR) spectroscopy. The results obtained were examined by univariate and multivariate data analysis (PCA, PLS-DA, and OPLS-DA). Metabolic profiling allowed us to quantify 43 resonance signals, of which 34 were identified. Multivariate statistical modeling revealed a highly significant separation between coma patients and brain-dead individuals, as well as strong predictive potential. The findings not only highlight the potential of the metabolomic approach for distinguishing patients in coma from those in the state of brain death but also may provide an understanding of the pathogenic mechanisms underlying these conditions.

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.

Surgical management of extracranial carotid artery aneurysms.

BACKGROUND: We present the methods and results of the surgical management of extracranial carotid artery aneurysms (ECCA). Postoperative complications including early and late neurological events were analysed. Correlation between reconstruction techniques and morphology of ECCA was assessed in this retrospective study. PATIENTS AND METHODS: In total, 32 reconstructions of ECCA were performed in 31 symptomatic patients with a mean age of 59.2 (range 33-84) years. The causes of ECCA were divided among atherosclerosis (n = 25; 78.1%), previous carotid endarterectomy with Dacron patch (n = 4; 12.5%), iatrogenic injury (n = 2; 6.3%) and infection (n = 1; 3.1%). In 23 cases, intervention consisted of carotid bypass. Aneurysmectomy with end-to-end suture was performed in 4 cases. Aneurysmal resection with patching was done in 2 cases and aneurysmorrhaphy without patching in another 2 cases. In 1 case, ligature of the internal carotid artery (ICA) was required. RESULTS: Technical success defined as the preservation of ICA patency was achieved in 31 cases (96.9%). There was one perioperative death due to major stroke (3.1%). Two cases of minor stroke occurred in the 30-day observation period (6.3%). Three patients had a transient hypoglossal nerve palsy that subsided spontaneously (9.4%). At a mean long-term follow-up of 68 months, there were no major or minor ipsilateral strokes or surgery-related deaths reported. In all 30 surviving patients (96.9%), long-term clinical outcomes were free from ipsilateral neurological symptoms. CONCLUSIONS: Open surgery is a relatively safe method in the therapy of ECCA. Surgical repair of ECCAs can be associated with an acceptable major stroke rate and moderate minor stroke rate. Complication-free long-term outcomes can be achieved in as many as 96.9 % of patients. Aneurysmectomy with end-to-end anastomosis or bypass surgery can be implemented during open repair of ECCA.