The majority of type 2 diabetic patients in Finnish primary care are at very high risk of cardiovascular events: A cross-sectional chart review study (STONE HF).

AIMS: To characterize clinical profiles, prevalence of chronic kidney disease (CKD), and treatment patterns in type 2 diabetes (T2D) and heart failure (HF) patients in Finnish primary care. METHODS: A total of 1385 patients (1196 with T2D, 50 with HF, and 139 with T2D and HF) in 60 Finnish primary care centers were recruited to this cross-sectional study. Data on demographic and clinical characteristics, laboratory measurements, and medications were collected retrospectively from medical records. T2D patients were classified according to their risk of cardiovascular (CV) events as very high-risk (62%) and other patients (38%). RESULTS: Of the T2D patients, 10% (139/1335) had a diagnosis of HF and 42% (457/1090) had stage 3-5 CKD and/or albuminuria based on laboratory measurement. Of the HF patients, 74% (139/189) had T2D and 78% (114/146) had stage 3-5 CKD and/or albuminuria. Metformin was the most frequently used medication in both very high-risk patients (74%) and other patients (86%). SGLT2 inhibitors and/or GLP-1 analogues were used by 37% of very high-risk patients compared to 42% in other patients. CONCLUSIONS: The majority of T2D patients in Finnish primary care are at very high risk of cardiovascular events. However, the implementation of treatments with proven cardioprotective effects in very high-risk patients is currently suboptimal.

Adenoviral VEGF-DΔN ΔC gene therapy for myocardial ischemia.

Background: Cardiovascular diseases are the leading cause of death globally. In spite of the availability of improved treatments, there is still a large group of chronic ischemia patients who suffer from significant symptoms and disability. Thus, there is a clear need to develop new treatment strategies for these patients. Therapeutic angiogenesis is a novel therapy method which has shown promising results in preclinical studies. In this study, we evaluated safety and efficacy of adenoviral (Ad) VEGF-DΔNΔC gene transfer for the treatment of myocardial ischemia in a pig model. Methods: Adenoviral VEGF-DΔNΔC gene transfer was given to pigs (n = 26) via intramyocardial injections using an electromechanical injection catheter. Angiogenic effects were evaluated in an acute myocardial infarction model (n = 18) and functionality of the lymphatic vessels were tested in healthy porcine myocardium (n = 8). AdLacZ was used as a control. Results: AdVEGF-DΔNΔC induced safe and effective myocardial angiogenesis by inducing a four-fold increase in mean capillary area at the edge of the myocardial infarct six days after the gene transfer relative to the control AdLacZ group. The effect was sustained over 21 days after the gene transfer, and there were no signs of vessels regression. AdVEGF-DΔNΔC also increased perfusion 3.4-fold near the infarct border zone relative to the control as measured by fluorescent microspheres. Ejection fraction was 8.7% higher in the AdVEGF-DΔNΔC treated group 21 days after the gene transfer relative to the AdLacZ control group. Modified Miles assay detected a transient increase in plasma protein extravasation after the AdVEGF-DΔNΔC treatment and a mild accumulation of pericardial effusate was observed at d6. However, AdVEGF-DΔNΔC also induced the growth of functional lymphatic vasculature, and the amount of pericardial fluid and level of vascular permeability had returned to normal by d21. Conclusion: Endovascular intramyocardial AdVEGF-DΔNΔC gene therapy proved to be safe and effective in the acute porcine myocardial infarction model and provides a new potential treatment option for patients with severe coronary heart disease.

Adenoviral VEGF-B186R127S gene transfer induces angiogenesis and improves perfusion in ischemic heart.

Vascular endothelial growth factor B (VEGF-B) is an interesting therapeutic candidate for coronary artery disease. However, it can also cause ventricular arrhythmias, potentially preventing its use in clinics. We cloned VEGF-B isoforms with different receptor binding profiles to clarify the roles of VEGFR-1 and Nrp-1 in angiogenesis and to see if angiogenic properties can be maintained while avoiding side effects. VEGF-B constructs were studied in vivo using adenovirus (Ad)-mediated intramyocardial gene transfers into the normoxic and ischemic porcine heart (n = 51). It was found that the unprocessed isoform VEGF-B186R127S is as efficient angiogenic growth factor as the native VEGF-B186 in normoxic and ischemic heart. In addition, AdVEGF-B186R127S increased myocardial perfusion reserve by 22% in ischemic heart without any side effects. AdVEGF-B127 (VEGFR-1 and Nrp-1 ligand) and AdVEGF-B109 (VEGFR-1 ligand) did not induce angiogenesis. Thus, VEGF-B186 is angiogenic only before its proteolytic processing to VEGF-B127. Only the VEGF-B186 C-terminal fragment was associated with arrhythmias.

Citrate-Saline-Formulated mRNA Delivery into the Heart Muscle with an Electromechanical Mapping and Injection Catheter Does Not Lead to Therapeutic Effects in a Porcine Chronic Myocardial Ischemia Model.

Based on recent success in using modified RNA in clinical applications, we tested the safety, feasibility, and efficacy of direct delivery of citrate-saline-formulated mRNA into an hibernating ischemic heart muscle using an electromechanical mapping and injection catheter system (NOGA/Myostar) in a porcine chronic myocardial ischemia model. Chronic ischemia was induced in domestic pigs (n = 24) using a bottleneck stent placed in the left anterior descending coronary artery. Low (1 mg) and high (7.5 mg) doses of citrate-saline-formulated vascular endothelial growth factor (VEGF)-A165 mRNA were administered in the study. LacZ mRNA and citrate-saline buffer were used as controls. Ten intramyocardial injections (200 µL each) of the mRNAs or citrate-saline buffer were given endovascularly into the hibernating ischemic myocardium using the NOGA catheter. Positron emission tomography 15O-radiowater imaging was performed 7 days after the induction of ischemia and 28 days after the mRNA delivery to measure quantitative myocardial blood perfusion. Coronary angiography, left ventricular function measurements, and clinical chemistry were obtained at each time point. Thirty-five days after the mRNA transfers, pigs were sacrificed, and infarct size and general histology were analyzed. LacZ mRNA pigs were sacrificed 24 h after the transduction. Citrate-saline-formulated mRNA delivery into the ischemic myocardium with endovascular injection catheter did not lead to meaningful transduction with the translation of VEGF-A165, nor therapeutic effects in the heart. VEGF-A165 mRNA showed no statistically significant improvements in left ventricular ejection fraction (LVEF), cardiac output, myocardial perfusion, infarct size, collateral growth, or capillary area in the study groups. However, there was a trend in the high-dose group toward an improved LVEF and cardiac output at rest. No significant adverse effects were observed. In conclusion, the NOGA/Myostar injection catheter system is ineffective in delivering citrate-saline-formulated mRNAs into the heart muscle with the doses and methods used in a porcine chronic myocardial ischemia model.

Ex Vivo Porcine Models Are Valid for Testing and Training Microsurgical Lumbar Decompression Techniques.

BACKGROUND: Spinal surgeries are the leading causes for patient settlement issues. Recent European Medical Device Regulations aims to reduce complications by enforcing that surgical tools are validated before clinical use. Human cadavers are favored in preclinical use, but due to anatomic variance, decay, and scarce supply, alternative synthetic and animal models are used. This study evaluates the fidelity and validity of porcine models in training and assessment of microsurgical decompressive techniques in the lumbar spine. METHODS: Anatomic dimensions of 10 human and 5 young pig spines were assessed from computed tomography images. Novel "en bloc" fresh-frozen ex vivo porcine model tissues' fidelity and validity for decompressive surgery was evaluated by 3 expert neurosurgeons, in comparison with other models. RESULTS: The pigs' anatomic dimensions were on average 11% smaller than in humans. The pigs' L4-L5 was most alike humans, and the highest similarity was in lamina and spinous process widths, and the skin to posterior longitudinal ligament distance. Dimensional variability was higher in humans (F = 19.06-0.56, P < 0.05). The pigs' tissues were felt as good as living patients and better than cadavers for skin, fascia, bone, facets, ligamentum flavum, and dura, but poor for vessels (experts' intraclass correlation coefficient = 0.696-0.903). The pig models' validity for assessing drills' adverse features (friction, jitter, heating, and soft tissue trauma) was reported to be unanimously excellent. CONCLUSION: Pigs are representative for assessing microsurgical decompression techniques in the lower lumbar spine. The novel "en bloc" pig model can be an asset for industries and clinicians during assessment and training of new spinal techniques.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing Myocardium.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4-5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Adenoviral Gene Transfer of Gremlin Modulates Vascular Endothelial Growth Factor-A-Induced Angiogenesis in Porcine Myocardium.

Coronary artery disease is a major cause of death and disability worldwide. New therapies are needed for patients who do not benefit or are not suitable for current treatments. Angiogenic gene therapy using vascular endothelial growth factors (VEGFs) has shown potential in preclinical trials. However, undesired side effects, such as increased permeability, limit their therapeutic potential. The aim of this study was to investigate if adenoviral gene transfer of a VEGF receptor 2 (VEGFR-2) ligand Gremlin, given simultaneously with VEGF-A, could modulate VEGFR-2-mediated increase in permeability without impairing the angiogenic effect of VEGF-A gene therapy. Gene transfers were done in pigs (n = 22) using endocardial injections with an endovascular injection catheter. Animals were divided in three groups receiving adenoviral (Ad) VEGF-A (n = 10), Gremlin (n = 6), or VEGF-A+Gremlin (n = 6) gene therapy. Animals were sacrificed and samples collected 6 days later for histological, safety, and permeability analyses. The mean capillary area was significantly increased in both treatment groups with AdVEGF-A when compared with the AdGremlin group. Also, the capillary area was significantly larger in AdVEGF-A group without AdGremlin. No significant differences in tissue permeability were observed using modified Miles assay between AdVEGF-A and AdVEGF-A+AdGremlin groups. However, cardiac tamponade and sudden cardiac deaths were observed only in the AdVEGF-A group. AdVEGF-A induces strong angiogenesis in porcine myocardium. Our results suggest that AdGremlin can limit the side effects of AdVEGF-A therapy, even though no direct effect on tissue permeability could be demonstrated. This could enable the use of larger AdVEGF-A doses to increase the treatment area and angiogenic effects without adverse side effects.

Isolation of fresh endothelial cells from porcine heart for cardiovascular studies: a new fast protocol suitable for genomic, transcriptomic and cell biology studies.

BACKGROUND: Endothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1-2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals. METHODS: Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig. RESULTS: The swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody. CONCLUSION: We developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.