Glycyrrhizic Acid Reduces Heart Rate and Blood Pressure by a Dual Mechanism.

Beta adrenergic receptors are crucial for their role in rhythmic contraction of heart along with their role in the pathological conditions such as tachycardia and high risk of heart failure. Studies report that the levels of beta-1 adrenergic receptor tend to decrease by 50%, whereas, the levels of beta-2 adrenergic receptor remains constant during the risk of heart failure. Beta blockers-the antagonistic molecules for beta-adrenergic receptors, function by slowing the heart rate, which thereby allows the left ventricle to fill completely during tachycardia incidents and hence helps in blood pumping capacity of heart and reducing the risk of heart failure. In the present study, we investigate the potential of glycyrrhizic acid (GA) as a possible principal drug molecule for cardiac arrhythmias owing to its ability to induce reduction in the heart rate and blood pressure. We use in vitro and in silico approach to study GA's effect on beta adrenergic receptor along with an in vivo study to examine its effect on heart rate and blood pressure. Additionally, we explore GA's proficiency in eliciting an increase in the plasma levels of vasoactive intestinal peptide, which by dilating the blood vessel consequently, can be a crucial aid during the occurrence of a potential heart attack. Therefore, we propose GA as a potential principal drug molecule via its potential in modulating heart rate and blood pressure.

Fucoidan and topography modification improved in situ endothelialization on acellular synthetic vascular grafts.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2 µm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2 µm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7 mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance.

In vivo evaluation of compliance mismatch on intimal hyperplasia formation in small diameter vascular grafts.

Small diameter synthetic vascular grafts have high failure rate due to the thrombosis and intimal hyperplasia formation. Compliance mismatch between the synthetic graft and native artery has been speculated to be one of the main causes of intimal hyperplasia. However, changing the compliance of synthetic materials without altering material chemistry remains a challenge. Here, we used poly(vinyl alcohol) (PVA) hydrogel as a graft material due to its biocompatibility and tunable mechanical properties to investigate the role of graft compliance in the development of intimal hyperplasia and in vivo patency. Two groups of PVA small diameter grafts with low compliance and high compliance were fabricated by dip casting method and implanted in a rabbit carotid artery end-to-side anastomosis model for 4 weeks. We demonstrated that the grafts with compliance that more closely matched with rabbit carotid artery had lower anastomotic intimal hyperplasia formation and higher graft patency compared to low compliance grafts. Overall, this study suggested that reducing the compliance mismatch between the native artery and vascular grafts is beneficial for reducing intimal hyperplasia formation.

Rabbit Surgery Protocol for End-to-End and End-to-Side Vascular Graft Anastomosis.

Preclinical testing in animal model is a required stage of vascular device development. Among small animal models, rabbits provide vasculature with relative larger caliber for anastomotic implantation of vascular grafts as preclinical testing before conducting large animal studies. Rabbits have similar hemostatic mechanism with human and can accommodate vascular grafts with various diameters at different locations, and thus provide a valid model to assess small-diameter vascular grafts. This chapter will describe the procedures and materials required to conduct survival surgery in rabbit carotid artery models for implantation of small-diameter tubular grafts with an end-to-side and end-to-end anastomotic technique.

Vascular Imaging in Small Animals Using Clinical Ultrasound Scanners.

Conventional ultrasound with frequency (2-15 MHz) has been a global diagnostic and therapeutic tool in clinical medicine, and high-frequency ultrasound (>30 MHz) has been a powerful investigative device for preclinical studies such as cardiovascular research. In this chapter, we describe the use of conventional ultrasound with a 2.5-10 MHz transducer as an investigative device for the measurement/detection of blood flow in rabbit model. The chapter will describe the procedures for the preparation of sonographer, imaging locations, and the details of the rabbits used as well as detailed imaging steps for the preoperative, immediately after operation, and postoperative follow-up ultrasound for vascular surgery, using a vascular graft implantation as an example. We also provide useful notes to avoid pitfalls for successful imaging. The overall goal of this chapter is to deliver the steps in using low-cost, non-invasive, and highly versatile clinical ultrasound imaging in preclinical small animal testing.

Enhanced efficiency of nonviral direct neuronal reprogramming on topographical patterns.

Nonviral direct neuronal reprogramming holds significant potential in the fields of tissue engineering and regenerative medicine. However, the issue of low reprogramming efficiency poses a major barrier to its application. We propose that topographical cues, which have been applied successfully to enhance lineage-directed differentiation and multipotent stem cell transdifferentiation, could improve nonviral direct neuronal reprogramming efficiency. To investigate, we used a polymer-BAM (Brn2, Ascl1, Myt1l) factor transfection polypex to reprogram primary mouse embryonic fibroblasts. Using a multiarchitecture chip, we screened for patterns that may improve transfection and/or subsequent induced neuron reprogramming efficiency. Selected patterns were then investigated further by analyzing ß-tubulin III (TUJ1) and microtubule-associated protein 2 (MAP2) protein expression, cell morphology and electrophysiological function of induced neurons. Certain hierarchical topographies, with nanopatterns imprinted on micropatterns, significantly improved the percentage of TUJ1+ and MAP2+ cells. It is postulated that the microscale base pattern enhances initial BAM expression while the nanoscale sub-pattern promotes subsequent maturation. This is because the base pattern alone increased expression of TUJ1 and MAP2, while the nanoscale pattern was the only pattern yielding induced neurons capable of firing multiple action potentials. Nanoscale patterns also produced the highest fraction of cells showing spontaneous synaptic activity. Overall, reprogramming efficiency with one dose of polyplex on hierarchical patterns was comparable to that of five doses without topography. Thus, topography can enhance nonviral direct reprogramming of fibroblasts into induced neurons.

Characterization and Functional Assessment of Endothelial Progenitor Cells in Ischemic Stroke Patients.

Endothelial dysfunction has been implicated in atherosclerosis, ischemic heart disease, and stroke. Endothelial progenitor cells (EPCs), found in the bone marrow and peripheral blood as rare cell population, demonstrated a high proliferation and differentiation capacity. Understanding how such diseases influence the quantity and functionality of EPCs is essential for the development of novel therapies. This study aims to investigate the factors that affect the quantity and functionality of circulating EPCs in stroke patients and healthy controls. Blood samples were collected once from healthy donors (n = 30) and up to 3 times (within 7 days (baseline), 3 and 12 months post-stroke) from stroke patients (n = 207). EPC subpopulations were isolated with flow cytometry for characterization. The Matrigel tubular formation assay was performed as a measure of functionality. An increased amount of circulating EPCs was observed in stroke patients over 45 years when compared to age-matched healthy individuals. EPCs showed a rising trend in stroke patients over the 12-month post-stroke period, reaching statistical significance at 12 months post-stroke. Isolated CD34+KDR+ cells from stroke patients showed impairment in tubular formation capability when compared to cells from healthy donors. The quantity and vasculogenic function of circulating EPCs in peripheral blood have been effectively evaluated in stroke patients and healthy control donors in this study. Age and stroke are found to be 2 influencing factors on the angiogenic capacity. It is suggested that the increase in EPC number is triggered by the recovery response following ischemic stroke. Graphical abstract.

Fucoidan functionalization on poly(vinyl alcohol) hydrogels for improved endothelialization and hemocompatibility.

The performance of clinical synthetic small diameter vascular grafts remains disappointing due to the fast occlusion caused by thrombosis and intimal hyperplasia formation. Poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties and a low thrombogenic surface, which suggests its potential value as a small diameter vascular graft material. However, PVA does not support cell adhesion and thus requires surface modification to encourage endothelialization. This study presents a modification of PVA with fucoidan. Fucoidan is a sulfated polysaccharide with anticoagulant and antithrombotic properties, which was shown to potentially increase endothelial cell adhesion and proliferation. By mixing fucoidan with PVA and co-crosslinked by sodium trimetaphosphate (STMP), the modification was achieved without sacrificing mechanical properties. Endothelial cell adhesion and monolayer function were significantly enhanced by the fucoidan modification. In vitro and ex-vivo studies showed low platelet adhesion and activation and decreased thrombin generation with fucoidan modified PVA. The modification proved to be compatible with gamma sterilization. In vivo evaluation of fucoidan modified PVA grafts in rabbits exhibited increased patency rate, endothelialization, and reduced intimal hyperplasia formation. The fucoidan modification presented here benefited the development of PVA vascular grafts and can be adapted to other blood contacting surfaces.

Effect of Ethylene Oxide Sterilization on Polyvinyl Alcohol Hydrogel Compared with Gamma Radiation.

This study investigated the effects of terminal sterilization of polyvinyl alcohol (PVA) biomaterials using clinically translatable techniques, specifically ethylene oxide (EtO) and gamma (γ) irradiation. While a few studies have reported the possibility of sterilizing PVA with γ-radiation, the use of EtO sterilization of PVA requires additional study. PVA solutions were chemically crosslinked with trisodium trimetaphosphate and sodium hydroxide. The three experimental groups included untreated control, EtO, and γ-irradiation, which were tested for the degree of swelling and water content, and mechanical properties such as radial compliance, longitudinal tensile, minimum bend radius, burst pressure, and suture retention strength. In addition, samples were characterized with scanning electron microscopy, differential scanning calorimetry, X-ray photoelectron spectroscopy, and water contact angle measurements. Cell attachment was assessed using the endothelial cell line EA.hy926, and the sterilized PVA cytotoxicity was studied with a live/dead stain. Platelet and fibrin accumulation was measured using an ex vivo shunt baboon model. Finally, the immune responses of PVA implants were analyzed after a 21-day subcutaneous implantation in rats and a 30-day implantation in baboon. EtO sterilization reduced the PVA graft wall thickness, its degree of swelling, and water content compared with both γ-irradiated and untreated PVA. Moreover, EtO sterilization significantly reduced the radial compliance and increased Young's modulus. EtO did not change PVA hydrophilicity, while γ-irradiation increased the water contact angle of the PVA. Consequently, endothelial cell attachment on the EtO-sterilized PVA showed similar results to the untreated PVA, while cell attachment significantly improved on the γ-irradiated PVA. When exposing the PVA grafts to circulating whole blood, fibrin accumulation of EtO-sterilized PVA was found to be significantly lower than γ-irradiated PVA. The immune responses of γ-irradiated PVA, EtO-treated PVA, and untreated PVA were compared. Implanted EtO-treated PVA showed the least MAC387 reaction. The terminal sterilization methods in this study changed PVA hydrogel properties; nevertheless, based on the characterizations performed, both sterilization methods were suitable for sterilizing PVA. We concluded that EtO can be used as an alternative method to sterilize PVA hydrogel material. Impact statement Polyvinyl alcohol (PVA) hydrogels have been used for a variety of tissue replacements, including neural, cardiac, meniscal, cartilage, muscle, pancreatic, and ocular applications. In addition, PVA can be made into a tubular shape and used as a small-diameter vascular graft. Ethylene oxide (EtO) is one of the Food and Drug Administration-approved methods for sterilization, but its effect on PVA has not been studied extensively. The outcome of this study provides the effects of EtO and γ-irradiation of PVA grafts on both the material properties and the in vivo responses, particularly for vascular applications. Knowledge of these effects may ultimately improve the success rate of PVA vascular grafts.

Loss of secretin results in systemic and pulmonary hypertension with cardiopulmonary pathologies in mice.

More than 1 billion people globally are suffering from hypertension, which is a long-term incurable medical condition that can further lead to dangerous complications and death if left untreated. In earlier studies, the brain-gut peptide secretin (SCT) was found to be able to control blood pressure by its cardiovascular and pulmonary effects. For example, serum SCT in patients with congestive heart failure was one-third of the normal level. These observations strongly suggest that SCT has a causal role in blood pressure control, and in this report, we used constitutive SCT knockout (SCT-/-) mice and control C57BL/6N mice to investigate differences in the morphology, function, underlying mechanisms and response to SCT treatment. We found that SCT-/- mice suffer from systemic and pulmonary hypertension with increased fibrosis in the lungs and heart. Small airway remodelling and pulmonary inflammation were also found in SCT-/- mice. Serum NO and VEGF levels were reduced and plasma aldosterone levels were increased in SCT-/- mice. Elevated cardiac aldosterone and decreased VEGF in the lungs were observed in the SCT-/- mice. More interestingly, SCT replacement in SCT-/- mice could prevent the development of heart and lung pathologies compared to the untreated group. Taken together, we comprehensively demonstrated the critical role of SCT in the cardiovascular and pulmonary systems and provide new insight into the potential role of SCT in the pathological development of cardiopulmonary and cardiovascular diseases.

Muscle-generated BDNF is a sexually dimorphic myokine that controls metabolic flexibility.

The ability of skeletal muscle to switch between lipid and glucose oxidation for ATP production during metabolic stress is pivotal for maintaining systemic energy homeostasis, and dysregulation of this metabolic flexibility is a dominant cause of several metabolic disorders. However, the molecular mechanism that governs fuel selection in muscle is not well understood. Here, we report that brain-derived neurotrophic factor (BDNF) is a fasting-induced myokine that controls metabolic reprograming through the AMPK/CREB/PGC-1α pathway in female mice. Female mice with a muscle-specific deficiency in BDNF (MBKO mice) were unable to switch the predominant fuel source from carbohydrates to fatty acids during fasting, which reduced ATP production in muscle. Fasting-induced muscle atrophy was also compromised in female MBKO mice, likely a result of autophagy inhibition. These mutant mice displayed myofiber necrosis, weaker muscle strength, reduced locomotion, and muscle-specific insulin resistance. Together, our results show that muscle-derived BDNF facilitates metabolic adaption during nutrient scarcity in a gender-specific manner and that insufficient BDNF production in skeletal muscle promotes the development of metabolic myopathies and insulin resistance.

BDNF mimetic alleviates body weight gain in obese mice by enhancing mitochondrial biogenesis in skeletal muscle.

BACKGROUND: 7,8-Dihydroxyflavone (7,8-DHF) is a small molecular weight compound that mimics the functions of brain-derived neurotrophic factor (BDNF). The current study aims to elucidate the molecular mechanism of 7,8-DHF-induced body weight regulation. METHODS: Obese female C57/BL6 (20-week-old) mice that have been fed with high-fat diet for 13 weeks were treated with 7,8-DHF for 9 weeks. Various biochemical and molecular analyses were performed to examine the signal transduction pathway, metabolite content, and mitochondrial mass in the animals. Moreover, systemic energy metabolism and insulin sensitivity were determined by indirect calorimetry and insulin/glucose-tolerance tests. We have also determined the metabolic actions of 7,8-DHF on cultured myotubes. RESULTS: 7,8-DHF treatment increased cellular respiration by promoting mitochondrial biogenesis in cultured skeletal muscle cells. In diet-induced obese mice, subsequent 7,8-DHF consumption triggered the AMPK/CREB/PGC-1α pathways to increase the muscular mitochondrial content. Systemic energy metabolism was thus elevated, which reduced the body weight gain in obese animals. Consequently, hyperlipidemia, hyperglycemia hyperinsulinemia, and ectopic lipid accumulation in skeletal muscle and liver of the obese animals were alleviated after 7,8-DHF treatment. Moreover, insulin sensitivity of the obese muscle was improved after 7,8-DHF consumption. CONCLUSION: 7,8-DHF treatment increases muscular mitochondrial respiration and systemic energy expenditure, which alleviates the body weight gain and partially reverse the metabolic abnormalities induced by obesity.

Minimally Invasive Transverse Aortic Constriction in Mice.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Tumor Necrosis Factor-α Promotes Phosphoinositide 3-Kinase Enhancer A and AMP-Activated Protein Kinase Interaction to Suppress Lipid Oxidation in Skeletal Muscle.

Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine that plays a central role in obesity-induced insulin resistance. It also controls cellular lipid metabolism, but the underlining mechanism is poorly understood. We report in this study that phosphoinositide 3-kinase enhancer A (PIKE-A) is a novel effector of TNF-α to facilitate its metabolic modulation in the skeletal muscle. Depletion of PIKE-A in C2C12 myotubes diminished the inhibitory activities of TNF-α on mitochondrial respiration and lipid oxidation, whereas PIKE-A overexpression exacerbated these cellular responses. We also found that TNF-α promoted the interaction between PIKE-A and AMP-activated protein kinase (AMPK) to suppress its kinase activity in vitro and in vivo. As a result, animals with PIKE ablation in the skeletal muscle per se display an upregulation of AMPK phosphorylation and a higher preference to use lipid as the energy production substrate under high-fat diet feeding, which mitigates the development of diet-induced hyperlipidemia, ectopic lipid accumulation, and muscle insulin resistance. Hence, our data reveal PIKE-A as a new signaling factor that is important for TNF-α-initiated metabolic changes in skeletal muscle.