Transmembrane stem factor nanodiscs enhanced revascularization in a hind limb ischemia model in diabetic, hyperlipidemic rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

A high throughput blood-brain barrier model incorporating shear stress with improved predictive power for drug discovery.

The blood-brain barrier is a key structure regulating the health of the brain and access of drugs and pathogens to neural tissue. Shear stress is a key regulator of the blood-brain barrier; however, the commonly used multi-well vitro models of the blood-brain barrier do not incorporate shear stress. In this work, we designed and validated a high-throughput system for simulating the blood-brain barrier that incorporates physiological flow and incorporates an optimized cellular model of the blood-brain barrier. This system can perform assays of blood-brain barrier function with shear stress, with 48 independent assays simultaneously. Using the high throughput assay, we conducted drug screening assays to explore the effects of compounds for opening or closing blood-brain barrier. Our studies revealed that assays with shear stress were more predictive and were able to identify compounds known to modify the blood-brain barrier function while static assays were not. Overall, we demonstrate an optimized, high throughput assay for simulating the blood-brain barrier that incorporates shear stress and is practical for use in drug screening and other high throughput studies of toxicology.

Syndecan-4 proteoliposomes enhance revascularization in a rabbit hind limb ischemia model of peripheral ischemia.

Regenerative therapeutics for treating peripheral arterial disease are an appealing strategy for creating more durable solutions for limb ischemia. In this work, we performed preclinical testing of an injectable formulation of syndecan-4 proteoliposomes combined with growth factors as treatment for peripheral ischemia delivered in an alginate hydrogel. We tested this therapy in an advanced model of hindlimb ischemia in rabbits with diabetes and hyperlipidemia. Our studies demonstrate enhancement in vascularity and new blood vessel growth with treatment with syndecan-4 proteoliposomes in combination with FGF-2 or FGF-2/PDGF-BB. The effects of the treatments were particularly effective in enhancing vascularity in the lower limb with a 2-4 increase in blood vessels in the treatment group in comparison to the control group. In addition, we demonstrate that the syndecan-4 proteoliposomes have stability for at least 28 days when stored at 4°C to allow transport and use in the hospital environment. In addition, we performed toxicity studies in the mice and found no toxic effects even when injected at high concentration. Overall, our studies support that syndecan-4 proteoliposomes markedly enhance the therapeutic potential of growth factors in the context of disease and may be promising therapeutics for inducing vascular regeneration in peripheral ischemia. STATEMENT OF SIGNIFICANCE: Peripheral ischemia is a common condition in which there is a lack of blood flow to the lower limbs. This condition can lead to pain while walking and, in severe cases, critical limb ischemia and limb loss. In this study, we demonstrate the safety and efficacy of a novel injectable therapy for enhancing revascularization in peripheral ischemia using an advanced large animal model of peripheral vascular disease using rabbits with hyperlipidemia and diabetes.

The effects of strain history on aortic valve interstitial cell activation in a 3D hydrogel environment.

Aortic valves (AVs) undergo unique stretch histories that include high rates and magnitudes. While major differences in deformation patterns have been observed between normal and congenitally defective bicuspid aortic valves (BAVs), the relation to underlying mechanisms of rapid disease onset in BAV patients remains unknown. To evaluate how the variations in stretch history affect AV interstitial cell (AVIC) activation, high-throughput methods were developed to impart varied cyclical biaxial stretch histories into 3D poly(ethylene) glycol hydrogels seeded with AVICs for 48 h. Specifically, a physiologically mimicking stretch history was compared to two stretch histories with varied peak stretch and stretch rate. Post-conditioned AVICs were imaged for nuclear shape, alpha smooth muscle actin (αSMA) and vimentin (VMN) polymerization, and small mothers against decapentaplegic homologs 2 and 3 (SMAD 2/3) nuclear activity. The results indicated that bulk gel deformations were accurately transduced to the AVICs. Lower peak stretches lead to increased αSMA polymerization. In contrast, VMN polymerization was a function of stretch rate, with SMAD 2/3 nuclear localization and nuclear shape also trending toward stretch rate dependency. Lower than physiological levels of stretch rate led to higher SMAD 2/3 activity, higher VMN polymerization around the nucleus, and lower nuclear elongation. αSMA polymerization did not correlate with VMN polymerization, SMAD 2/3 activity, nor nuclear shape. These results suggest that a negative feedback loop may form between SMAD 2/3, VMN, and nuclear shape to maintain AVIC homeostatic nuclear deformations, which is dependent on stretch rate. These novel results suggest that AVIC mechanobiological responses are sensitive to stretch history and provide insight into the mechanisms of AV disease.

Increased let-7d-5p in non-alcoholic fatty liver promotes insulin resistance and is a potential blood biomarker for diagnosis.

BACKGROUND AND AIMS: The molecular mechanisms driving non-alcoholic fatty liver disease (NAFLD) are poorly understood; however, microRNAs might play a key role in these processes. We hypothesize that let-7d-5p could contribute to the pathophysiology of NAFLD and serve as a potential diagnostic biomarker. METHODS: We evaluated let-7d-5p levels and its targets in liver biopsies from a cross-sectional study including patients with NAFLD and healthy donors, and from a mouse model of NAFLD. Moreover, the induction of let-7d-5p expression by fatty acids was evaluated in vitro. Further, we overexpressed let-7d-5p in vitro to corroborate the results observed in vivo. Circulating let-7d-5p and its potential as a NAFLD biomarker was determined in isolated extracellular vesicles from human plasma by RT-qPCR. RESULTS: Our results demonstrate that hepatic let-7d-5p was significantly up-regulated in patients with steatosis, and this increase correlated with obesity and a decreased expression of AKT serine/threonine kinase (AKT), insulin-like growth factor 1 (IGF1), IGF-I receptor (IGF1R) and insulin receptor (INSR). These alterations were corroborated in a NAFLD mouse model. In vitro, fatty acids increased let-7d-5p expression, and its overexpression decreased AKT, IGF-IR and IR protein expression. Furthermore, let-7d-5p hindered AKT phosphorylation in vitro after insulin stimulation. Finally, circulating let-7d-5p significantly decreased in steatosis patients and receiver operating characteristic (ROC) analyses confirmed its utility as a diagnostic biomarker. CONCLUSIONS: Our results highlight the emerging role of let-7d-5p as a potential therapeutic target for NAFLD since its overexpression impairs hepatic insulin signalling, and also, as a novel non-invasive biomarker for NAFLD diagnosis.

Transmembrane Stem Factor Nanodiscs Enhanced Revascularization in a Hind Limb Ischemia Model in Diabetic, Hyperlipidemic Rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

Mechanical conditioning of human mesenchymal stem cells for enhancing vascular regeneration.

Human mesenchymal stem cells (hMSCs) are an appealing cell type for therapeutic applications but remain limited by poor efficacy in clinical trials. Here, we describe a conditioning technique that enhances the vascular regenerative properties of hMSCs and increases their expression of endothelial cell and pericyte markers. We also describe an alginate gel encapsulation protocol for delivering the conditioned cells. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).1.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation.

Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

Optimized design of a hyperflexible sieve electrode to enhance neurovascular regeneration for a peripheral neural interface.

One in 190 Americans is currently living with the loss of a limb resulted from injury, amputation, or neurodegenerative disease. Advanced neuroprosthetic devices combine peripheral neural interfaces with sophisticated prosthetics and hold great potential for the rehabilitation of impaired motor and sensory functions. While robotic prosthetics have advanced very rapidly, peripheral neural interfaces have long been limited by the capability of interfacing with the peripheral nervous system. In this work, we developed a hyperflexible regenerative sieve electrode to serve as a peripheral neural interface. We examined tissue neurovascular integration through this novel device. We demonstrated that we could enhance the neurovascular invasion through the device with directional growth factor delivery. Furthermore, we demonstrated that we could reduce the tissue reaction to the device often seen in peripheral neural interfaces. Finally, we show that we can create a stable tissue device interface in a long-term implantation that does not impede the normal regenerative processes of the nerve. Our study developed an optimal platform for the continued development of hyperflexible sieve electrode peripheral neural interfaces.

Molecular tension in syndecan-1 is regulated by extracellular mechanical cues and fluidic shear stress.

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.

Biomechanical regulation of breast cancer metastasis and progression.

Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3Kγ inhibitor.

Laser nanobubbles induce immunogenic cell death in breast cancer.

Recent advances in immunotherapy have highlighted a need for therapeutics that initiate immunogenic cell death in tumors to stimulate the body's immune response to cancer. This study examines whether laser-generated bubbles surrounding nanoparticles ("nanobubbles") induce an immunogenic response for cancer treatment. A single nanosecond laser pulse at 1064 nm generates micron-sized bubbles surrounding gold nanorods in the cytoplasm of breast cancer cells. Cell death occurred in cells treated with nanorods and irradiated, but not in cells with irradiation treatment alone. Cells treated with nanorods and irradiation had increased damage-associated molecular patterns (DAMPs), including increased expression of chaperone proteins human high mobility group box 1 (HMGB1), adenosine triphosphate (ATP), and heat shock protein 70 (HSP70). This enhanced expression of DAMPs led to the activation of dendritic cells. Overall, this treatment approach is a rapid and highly specific method to eradicate tumor cells with simultaneous immunogenic cell death signaling, showing potential as a combination strategy for immunotherapy.

Mechanobiological conditioning of mesenchymal stem cells for enhanced vascular regeneration.

Using endogenous mesenchymal stem cells for treating myocardial infarction and other cardiovascular conditions typically results in poor efficacy, in part owing to the heterogeneity of the harvested cells and of the patient responses. Here, by means of high-throughput screening of the combinatorial space of mechanical-strain level and of the presence of particular kinase inhibitors, we show that human mesenchymal stem cells can be mechanically and pharmacologically conditioned to enhance vascular regeneration in vivo. Mesenchymal stem cells conditioned to increase the activation of signalling pathways mediated by Smad2/3 (mothers against decapentaplegic homolog 2/3) and YAP (Yes-associated protein) expressed markers that are associated with pericytes and endothelial cells, displayed increased angiogenic activity in vitro, and enhanced the formation of vasculature in mice after subcutaneous implantation and after implantation in ischaemic hindlimbs. These effects were mediated by the crosstalk of endothelial-growth-factor receptors, transforming-growth-factor-beta receptor type 1 and vascular-endothelial-growth-factor receptor 2. Mechanical and pharmacological conditioning can significantly enhance the regenerative properties of mesenchymal stem cells.

A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression.

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

Genome wide analysis of gene expression changes in skin from patients with type 2 diabetes.

Non-healing chronic ulcers are a serious complication of diabetes and are a major healthcare problem. While a host of treatments have been explored to heal or prevent these ulcers from forming, these treatments have not been found to be consistently effective in clinical trials. An understanding of the changes in gene expression in the skin of diabetic patients may provide insight into the processes and mechanisms that precede the formation of non-healing ulcers. In this study, we investigated genome wide changes in gene expression in skin between patients with type 2 diabetes and non-diabetic patients using next generation sequencing. We compared the gene expression in skin samples taken from 27 patients (13 with type 2 diabetes and 14 non-diabetic). This information may be useful in identifying the causal factors and potential therapeutic targets for the prevention and treatment of diabetic related diseases.

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

Therapeutic strategies for enhancing angiogenesis in wound healing.

The enhancement of wound healing has been a goal of medical practitioners for thousands of years. The development of chronic, non-healing wounds is a persistent medical problem that drives patient morbidity and increases healthcare costs. A key aspect of many non-healing wounds is the reduced presence of vessel growth through the process of angiogenesis. This review surveys the creation of new treatments for healing cutaneous wounds through therapeutic angiogenesis. In particular, we discuss the challenges and advancement that have been made in delivering biologic, pharmaceutical and cell-based therapies as enhancers of wound vascularity and healing.

Algal Polysaccharides as Therapeutic Agents for Atherosclerosis.

Seaweed-derived polysaccharides including agar and alginate, have found widespread applications in biomedical research and medical therapeutic applications including wound healing, drug delivery, and tissue engineering. Given the recent increases in the incidence of diabetes, obesity and hyperlipidemia, there is a pressing need for low cost therapeutics that can economically and effectively slow the progression of atherosclerosis. Marine polysaccharides have been consumed by humans for millennia and are available in large quantities at low cost. Polysaccharides such as fucoidan, laminarin sulfate and ulvan have shown promise in reducing atherosclerosis and its accompanying risk factors in animal models. However, others have been tested in very limited context in scientific studies. In this review, we explore the current state of knowledge for these promising therapeutics and discuss the potential and challenges of using seaweed derived polysaccharides as therapies for atherosclerosis.

Recruitment and therapeutic application of macrophages in skeletal muscles after hind limb ischemia.

OBJECTIVE: Peripheral arterial disease can cause not only ischemia but also skeletal muscle damage. It has been known that macrophages (MPs) play an important role in coordinating muscle repair; however, phenotype transition of monocyte-MP in ischemic muscle has not been well defined. Hence, the purpose of this study was to examine the temporal recruitment of MPs and to explore their therapeutic effect on ischemic muscle regeneration. METHODS: Unilateral femoral artery excision was performed on C57BL/6 mice. Myeloid cells were isolated from the ischemic muscles, characterized using flow cytometry. Bone marrow-derived MPs were injected (2 × 106 cells) into the ischemic gastrocnemius muscle 24 hours after injury. Blood flow recovery was measured using laser speckle imaging. Functional outcome was evaluated by assessing the contractile force of ischemic muscles. Histologic analysis included quantification of myofiber size, collagen deposition, number of inflammatory and MyoD-expressing cells, and capillary density. RESULTS: Neutrophils and inflammatory monocytes-MPs were present at day 1 after injury. The mature MPs then remained elevated as the dominant population from day 5 to day 21 with the observation of regenerating fibers. Functional measurements revealed that the force production was significantly enhanced after treatment with proinflammatory M1 MPs (94.9% vs 77.9%; P < .05), and this was consistent with increased myofiber size, capillary- fiber ratio, and perfusion (78.6% vs 39.9%; P < .05). Moreover, the percentage of MyoD-expressing nuclei was significantly higher at day 4, indicating that M1 MPs may hasten muscle repair. Whereas early delivery of anti-inflammatory M2 MPs improved myofiber size, this was accompanied by persistent fibrosis suggesting ongoing tissue remodeling, and lower force production was observed. CONCLUSIONS: We demonstrated the dynamics of myeloid cells in skeletal muscle after ischemic insult, and the administration of exogenous M1 MPs in a temporally coordinated manner successfully improved angiogenesis and skeletal muscle regeneration. Our results suggested that cell therapy using MPs may be a promising adjunctive therapeutic approach for peripheral arterial disease.