Browsing by Author "He, Jia-Qiang"

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    Aberrant hippocampal neurogenesis contributes to learning and memory deficits in a mouse model of repetitive mild traumatic brain injury
    Greer, Kisha (Virginia Tech, 2019-10-02)
    Adult hippocampal neurogenesis, or the process of creating new neurons in the dentate gyrus (DG) of the hippocampus, underlies learning and memory capacity. This cognitive ability is essential for humans to operate in their everyday lives, but cognitive disruption can occur in response to traumatic insult such as brain injury. Previous findings in rodent models have characterized the effect of moderate traumatic brain injury (TBI) on neurogenesis and found learning and memory shortfalls correlated with limited neurogenic capacity. While there are no substantial changes after one mild TBI, research has yet to determine if neurogenesis contributes to the worsened cognitive outcomes of repetitive mild TBI. Here, we examined the effect of neurogenesis on cognitive decline following repetitive mild TBI by utilizing AraC to limit the neurogenic capacity of the DG. Utilizing a BrdU fate-labeling strategy, we found a significant increase in the number of immature neurons that correlate learning and memory impairment. These changes were attenuated in AraC-treated animals. We further identified endothelial cell (EC)-specific EphA4 receptor as a key mediator of aberrant neurogenesis. Taken together, we conclude that increased aberrant neurogenesis contributes to learning and memory deficits after repetitive mild TBI.
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    Chronic Treatment of TMAO Undermines Mouse Cardiac Structure and Function in a Sex-specific Manner
    Ding, Hanzhang (Virginia Tech, 2023-12-19)
    Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, with adverse outcomes of CVD. As of today, TMAO's effects on cardiac structure and function are not well understood. In this study, both male and female TMAO-treated hearts showed functional deficits based on electrocardiography and echocardiography results. Immunohistochemistry results showed signs of hypertrophic cardiomyopathy in TMAO-treated male hearts while female TMAO-treated hearts showed signs of dilated cardiomyopathy. Neither TMAO group showed signs of fibrosis. Overproduction of reactive oxygen species was only observed in male TMAO-treated hearts. At the level of individual cardiomyocytes, significant delays in time to reach maximum contraction and dilation were only seen in TMAO-treated male hearts along with higher contractile force. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to comprehensively characterize features of TMAO-induced heart failure in both males and females which extends our current knowledge from human clinical associations.
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    Common differentially expressed genes and pathways correlating both coronary artery disease and atrial fibrillation
    Zheng, Youjing; He, Jia-Qiang (2021-05-03)
    Coronary artery disease (CAD) and atrial fibrillation (AF) share common risk factors, such as hypertension and diabetes. The patients with CAD often suffer concomitantly AF, but how two diseases interact with each other at cellular and molecular levels remain largely unknown. The present study aims to dissect the common differentially expressed genes (DEGs) that are concurrently associated with CAD and AF. Two datasets [GSE71226 for CAD) and GSE31821 for AF] were analyzed with GEO2R and Venn Diagram to identify the DEGs. Signaling pathways, gene enrichments, and protein-protein interactions (PPI) of the identified common DEGs were further analyzed with Kyoto Encyclopedia of Gene and Genome (KEGG), Database for Annotation, Visualization and Integrated Discovery (DAVID), and Search Toll for the Retrieval of Interacting Genes (STRING). 565 up- and 1367 down-regulated genes in GSE71226 and 293 up- and 68 down-regulated genes in GSE31821 were identified. Among those, 21 common DEGs were discovered from both datasets, which lead to the findings of 4 CAD and 21 AF pathways, 3 significant gene enrichments (intracellular cytoplasm, protein binding, and vascular labyrinthine layer), and 3 key proteins (membrane metallo-endopeptidase (MME), transferrin receptor 1 (TfR1), and Lyso-some-associated membrane glycoprotein 1 (LAMP1)). Together, these data implied that these three proteins may play a central role in development of both CAD and AF.
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    Conversion of equine umbilical cord matrix mesenchymal stem cells to the trophectoderm lineage using the Yamanaka reprogramming factors
    Reinholt, Brad M. (Virginia Tech, 2015-07-21)
    Induced pluripotent stem (iPS) cells that possess embryonic stem (ES) cell-like properties are generated through the use of the Yamanaka transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). Advanced transgene delivery methods utilizing non-integrating viruses for transduction of target cells has provided new opportunities for regenerative medicine in humans and other species. We sought to use this technology to generate equine iPS cells to address challenges in equine regenerative medicine. Umbilical cord matrix mesenchymal stromal cells (MSC) were transduced with the non-integrating Sendai virus encoding for the OSKM transcription factors. The cells initially were cultured on mouse embryonic feeder cells supplemented with LIF (10 ng/mL) and FGF2 (4 ng/mL). Transduction generated 21 initial colonies. Of these, four survived beyond 20 passages. The transduced equine cells morphologically resembled ES cells and expressed cell surface antigens indicative of ES cells. Molecular evaluation revealed the cells maintained expression of endogenous OSKM while the exogenous OSK transgenes were extinguished, but MYC was maintained. The transduced equine cells did not express the ES marker NANOG, but did express the trophectoderm markers CDX2 and TFAP2A. Both OCT4 and CDX2 were colocalized to the nucleus. The transduced equine cells were termed equine induced trophoblast (iTr) cells. Culture of the iTr cell in suspension resulted in formation of blastocyst-like spheres rather than solid cell aggregates indicative of ES and iPS cells. The iTr cells were transitioned to a feeder free monolayer culture. Transformation of the iTr cells to the spherical arrangement stimulated expression of genes that mark differentiation of trophoblast cells and up-regulated 250 transcripts over the monolayer arrangement. The iTr monolayer arrangement up-regulated 50 transcripts compared to the spherical arrangement. The iTr spheres respond to BMP4, EGF, and FGF2 by phosphorylating signal transduction proteins. Addition of BMP4, EGF, or FGF2 in combined pairs was able to alter TFAP2A, NEU1, and SLC35A1 expression. The generation of iTr cells by transduction of the Yamanaka reprogramming factors is not unique to equine cells. However, this report marks the generation of the first equine trophoblast cell line capable of recapitulating early equine trophoblast development. The new iTr line could prove valuable in gaining greater understanding of equine trophectoderm development.
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    The Direct Impact of Trimethelamine-N-Oxide on Cardiac Function
    Zheng, Youjing (Virginia Tech, 2023-02-15)
    Cardiovascular diseases (CVDs) are the leading cause of death and disability worldwide. The aging population and the rapidly increasing prevalence of obesity and type 2 diabetes will contribute to a growing epidemic of CVDs globally. Despite the extensive investigations in etiology, the pathogenesis of CVDs still not fully understand, and the treatment and prevention for CVDs are still limited. Significant interest has been raised in gut microbiota-host interaction since increasing evidence revealed that gut microbiomes play an important role in human health and diseases, including CVDs. Among more than two thousand gut microbiota metabolites, a compound named trimethylamine N-oxide (TMAO) was revealed to be closely related to CVDs. However, the impact of TMAO on cardiovascular health is still full of controversy and the direct impact of TMAO on heart tissue and cardiomyocytes has not been fully understood yet. In the first chapter, we reviewed the literature on TMAO-related atherosclerosis and cardiomyopathy to give us a general aspect of current research progress in the role of TMAO on CVDs. In this context, we provide an overview of the potential mechanisms underlying TMAO-induced cardiovascular diseases at the cellular and molecular levels, with a focus on atherosclerosis and cardiomyopathy. We also address the direct effects of TMAO on cardiomyocytes (a new and under-researched area) and finally propose TMAO as a potential biomarker and/or therapeutic target for the diagnosis and treatment of patients with CVDs. In the second chapter, the direct impact of TMAO on cardiac function was tested in vivo using wild-type C57B6L mice model. Four experiment groups were enrolled in the feeding protocol, which included 3w (different time points), 6w, and 13w feeding time to reveal the impact of short and longer periods of TMAO consumption on cardiac function. The plasma TMAO was measured by liquid chromatography-tandem mass spectrometry (LC/MS/MS) method at the end of the feeding protocol. Echocardiography and electrocardiography (ECG) were performed to assess the overall heart function. The histopathology staining was used to evaluate the cardiac microstructure change. By the end of the feeding protocol, the plasma TMAO all increased significantly in the TMAO group compared to the control no matter the TMAO feeding period. Echocardiography showed that 6w and 13w TMAO intake could significantly decrease cardiac contractility evidenced by decreased eject fraction (EF) and fraction shortening (FS). The electrocardiography (ECG) showed decreased R wave aptitude in 6w and 13w TMAO feed group with sinus rhythm. However, 3w TMAO intake had no impact on both cardiac contractability and ECG. Moreover, chronic TMAO supplement (13w) showed increased left ventricle (LV) mass on echocardiography and increased LV thickness on the tissue section. Further histology analysis revealed cardiomyocyte hypertrophy in the 13w TMAO-treated male group. Notably, the female mice showed significantly higher TMAO levels both in the control and treated group compared to the male, however, no gender difference was observed as to the ECG and echocardiography. In addition, the plasma inflammation cytokines were also analyzed and the tumor necrosis factor-α (TNF- α), interleukin 10 (IL-10), Fibroblast growth factor 2 (FGF β) and leptin were all increased in the 13w TMAO treated group compared to the control. These results suggest that chronic TMAO exposure led to increased plasma TMAO levels, which contribute to system inflammation and cardiac dysfunction due to cardiac hypertrophy in mice models. Research in chapter 3 demonstrates the potential underlying mechanisms of TMAO-induced cardiac dysfunction using adult mouse cardiomyocytes. In this study, we examined the direct effect of TMAO on reactive oxidative species (ROS) generation and factors related to cardiomyocyte contractibility, including, microtubule, Connexin43 (Cx43) expression, and gap junction intracellular communication (GJIC), intracellular calcium dynamics and transversal-tubule (T-tubule) both in acute and chronic TMAO challenge. Moreover, we also tested whether TMAO can enter cardiomyocytes directly. The results suggested that TMAO could enter cardiomyocytes through organic cation transporters (OCTs) and promote increased ROS generation via augmentation of NADPH oxidase 4 (Nox4). Moreover, both acute and chronic TMAO exposure could induce microtubule densification, which plays a critical role in intracellular protein transportation and cardiomyocyte morphology maintenance. We also demonstrated chronic TMAO exposure could inhibit the Cx43 expression at both cellular and tissue level, and therefore impact the GJIC for the first time. Besides, we also revealed that TMAO could interrupt intracellular calcium handling both acutely and chronically, especially documented by decreased efficiency in intracellular calcium removal, related to decreased sarcoplasmic reticulum Ca2+-ATPase (Serca2) expression. However, TMAO showed no impact on cardiomyocyte T-tubule network organization. Taken together, we demonstrated a direct destructive role of TMAO on cardiomyocytes' functional properties and provided a novel potential mechanism for TMAO-induced cardiac dysfunction. Overall, the research in this dissertation demonstrated the direct impact of TMAO on cardiomyocytes and cardiac function both in vivo and in vitro and evaluated the effect of TMAO both acutely and chronically. The TMAO can enter cardiomyocytes and induce Nox4-mediated oxidative stress, which could connect to multiple intracellular pathways, including microtubule densification, decreased Cx43 expression, and GJIC, as well as calcium handling dysfunction. Meanwhile, all these changes were closely related to the cardiomyocyte swelling observed in mice cardiac tissue after chronic TMAO consumption, which could ultimately contribute to cardiac contractile dysfunction and electrophysiology change in mice models.
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    Effects of Trimethylamine N-Oxide on Mouse Embryonic Stem Cell Properties
    Barron, Catherine Mary (Virginia Tech, 2020-08-06)
    Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity.
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    Endothelial Cell-Specific Knockout of Meis1 Protects Ischemic Hindlimb Through Vascular Remodeling
    Chen, Miao (Virginia Tech, 2018-06-28)
    Peripheral artery disease (PAD) affects more than 200 million people worldwide. PAD refers to illness due to a reduction or complete occlusion of blood flow in the artery, especially to the extremities in disease conditions, such as atherosclerosis or diabetes. Critical limb ischemia (CLI) is a severe form of PAD associated with high morbidity and mortality. Currently, no effective and permanent treatments are available for this disease. The current endovascular medications (e.g., angioplasty or stents) only relieve the clinical symptoms while the surgical therapies (e.g., bypass or endarterectomy) require grafting vessels from a healthy organ to the diseased limb of the patient. However, even with these therapeutic techniques, 30% of patients still undergo limb amputation within a year. Thus, understanding of disease mechanism and development of new therapeutic approaches are in urgent needs. Meis1 (myeloid ecotropic viral integration site 1) gene belongs to the three-amino-acid loop extension subclass of homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. Up to date, little is known about the role of Meis1 in regulating vascular remodeling under ischemic condition. In this study, we aim to investigate the role and underlying mechanism of Meis1 in the regulation of arteriogenesis and angiogenesis using hindlimb ischemia model of transgenic neonatal mice. The long-term goal is to develop a new treatment for patients with PAD. Three separate but related studies were planned to complete the proposed research aims. To better understand the role of Meis1, we reviewed, in the first chapter, all literature relevant to the recent advances of the Meis1 in normal hematopoiesis, vasculogenesis, and heart developments, which were mostly studied in zebrafish and mouse. Briefly, Meis1 is found to be highly expressed in the brain and retina in zebrafish and additional in the heart, nose, and limb in mouse during the very early developmental stage, and remains at a low level quickly after birth. Meis1 is necessary for both primitive and definitive hematopoiesis and required for posterior erythroid differentiation. The absence of Meis1 results in a severe reduction of the number of mature erythrocytes and weakens the heart beats in zebrafish. Meis1 deficiency mouse is dead as early as E11.5 due to the severe internal hemorrhage. In addition, Meis1 is essential in heart development. Knock-down of Meis1 can promote angiotensin II-induced cardiomyocytes (CMs) hypertrophy or CMs proliferation, which can be repressed by a transcription factor Tbx20. Meis1 appears to play a complicated role in the blood vessels. Although the major blood vessels are still normal when global deletion of Meis1, the intersegmental vessel cannot be formed in Meis1 morphants in the zebrafish, and the small vessels are either too narrow or form larger sinuses in Meis1 deficient mouse. The effects of Meis1 on the vascular network under normal and disease (ischemia) condition remain largely unknown, and the existing data in this field is limited. In the second chapter, we developed a method protocol to identify mice of all ages, especially neonates that we faced methodological difficulties to easily and permanently label prior to our major experiments. In this study, single- or 2-color tattooing (ear, tail, or toe or combinations) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day (PND) 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than three weeks. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. Thus, tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals, which shows a new approach than any existing method to identify mouse at all ages, especially the neonatal pups used in the present study (Chapter 4). Various formation of hindlimb ischemia with ligations of femoral artery or vein or both have been reported in the literature. The ischemic severity varies dependent on mouse strains and ligation methods. Due to the tiny body size of our experimental neonatal mice (PND2), it is technically challenging to separate the femoral artery from femoral vein without potential bleeding. In the third chapter, we aimed to explore a suitable surgical approach that can apply to neonatal mice. To this end, we compared the effects of femoral artery/vein (FAV) excision vs. femoral artery (FA) excision on hindlimb model using adult CD-1 mice. We showed during the 4-week period of blood reperfusion, no statistically significant differences were found between FAV and FA excision-induced ischemia regarding the reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell size. We conclude that FAV and FA excision in CD-1 mice generate a comparable severity of hindlimb ischemia. In other words, FAV ligation is no more severe than FA ligation. These findings provide valuable information for researchers when selecting ligation methods for their neonate hindlimb models. Based on these findings, we selected FAV ligation of hindlimb ischemia approach to study the function of Meis1 in vascular remodeling of neonatal mice. In the fourth chapter (the main part of my dissertation), we investigated the roles of Meis1 in regulating arteriogenesis and angiogenesis of neonatal mouse under the ischemic condition. To this end, endothelial cell-specific deletion of Meis1 was generated by cross-breeding Meis1flox/flox mice with Tie2-Cre mice. Wild-type (WT, Meis1f/f) and endothelial cell-specific knock-out (KO, Meis1ec-/-Tie2-Cre+) C57BL/6 mice at the age of PND2 were used. Under the anesthesia, the pups were subject to hindlimb ischemia by excising FAV. Laser Doppler Imager was used to measure the blood flow pre- and post-surgery up to 28 days. Toe necrosis, skeletal regeneration, and vascular distributions were examined at the end of experiments (PND28 post-ischemia). Surprisingly, during 4-week periods after ischemia, the blood flow ratios (ischemic vs. control limb) in KO mice significantly increased compared to WT on PND14 and PND28, suggesting the inhibitory effects of Meis1 on blood flow recovery under ischemic condition. Meanwhile, WT mice showed more severe necrotic limb (lower ratio of limb length and area, and higher necrotic scores at PND7) than those in the KO mice. Furthermore, significant increases in diameters of Dil-stained arterioles of the skin vessel and the vessels on the ligation site were observed in KO mice, indicating the enhanced arteriogenesis in KO mice. To investigate the underlying mechanism, RNA from the ischemia and control limb was extracted and q-PCR was used to study the potential genes involved in the mechanism. Casp3 and Casp8 were found downregulated showing less apoptosis in the KO mice. On the other hand, endothelial cells (ECs) were isolated from the lungs of 3-5 WT and KO neonates using CD31 Microbeads. CD31+ cells were plated and treated with 0, 0.5, and 1μM doxorubicin for 24 hours and analyzed with various assays. Meis1-KO ECs demonstrated higher cell viability and formed a higher number of vascular tubes than those in WT ECs following 0.5μM Dox treatment, presenting the potential ability of angiogenesis in KO-ECs. Furthermore, the increased viability in KO ECs may be due to the decreased expression or activities of Casp8 and Casp3. In conclusion, my present studies have developed a new methodology to easily and permanently identify all mice at any ages. The insignificant differences between FAV and FA ligations suggest that a relative-easy surgical approach could be used to generate hindlimb ischemic model, which potentially reduces the cost, decreases the surgical time and prevents damage of femoral nerve from surgical tools. More importantly, by using transgenic mice, we found that Meis1-KO dramatically increased blood flow and protected the ischemic hindlimb through vascular remodeling. Obviously, the molecular and cellular mechanisms underlying the above beneficial effects appear complicated and likely to involve multiple cellular remodeling processes and molecular signaling pathways to enhance arteriogenesis and angiogenesis and/or reduce cellular apoptosis through Meis1-mediated pathways. Our study demonstrated that under ischemic condition, knockout of Meis1 increases expression of Hif1a, which then activates Agt or VEGF, thus enhances arteriogenesis or angiogenesis; In addition, knockout of Meis1 activates Ccnd1, which subsequently promotes regeneration of skeletal muscle, and reduces expression of Casp8 and Casp3, thus preventing limb tissue from ischemia-induced apoptosis. Our innovative findings offer great potential to ultimately lead to new drug discovery or therapeutic approaches for prevention or treatment of PAD.
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    Endothelial-Specific EphA4 Negatively Regulates Native Pial Collateral Formation and Re-Perfusion following Hindlimb Ischemia
    Okyere, Benjamin; Giridhar, Kaavya; Hazy, Amanda; Chen, Miao; Keimig, David; Bielitz, Robert C.; Xie, Hehuang; He, Jia-Qiang; Huckle, William R.; Theus, Michelle H. (PLOS, 2016-07-28)
    Leptomeningeal anastomoses play a critical role in regulating vascular re-perfusion following obstruction, however, the mechanisms regulating their development remains under investingation. Our current findings indicate that EphA4 receptor is a novel negative regulator of collaterogenesis. We demonstrate that EphA4 is highly expressed on pial arteriole collaterals at post-natal day (P) 1 and 7, then significantly reduced by P21. Endothelial cell (EC)-specific loss of EphA4, EphA4f/f/Tie2::Cre (KO), resulted in an increase in the density but not diameter of pial collaterals compared to WT mice. ECs isolated from KO mice displayed a 3-fold increase in proliferation, enhanced migration, tube formation and elevated levels of phospho(p)-Akt compared to WT ECs. Attenuating p-Akt, using LY294002, reduced the proliferative and migration effects in the KO ECs. RNAseq analysis also revealed altered expression patterns for genes that regulate cell proliferation, vascular development, extracellular matrix and immune-mediate responses, namely MCP-1, MMP2 and angiopoietin-1. Lastly, we show that induction of hindlimb ischemia resulted in accelerated re-perfusion, collateral remodeling and reduced tissue necrosis in the absence of ECspecific EphA4 compared to WT mice. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and suggests a role in regulating vascular remodeling after obstruction.
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    In vivo degradation forms, anti-degradation strategies, and clinical applications of therapeutic peptides in non-infectious chronic diseases
    Tasdemiroglu, Yagmur; Gourdie, Robert G.; He, Jia-Qiang (Elsevier, 2022-10-15)
    Current medicinal treatments for diseases comprise largely of two categories: small molecular (chemical) (e.g., aspirin) and larger molecular (peptides/proteins, e.g., insulin) drugs. Whilst both types of therapeutics can effectively treat different diseases, ranging from well-understood (in view of pathogenesis and treatment) examples (e.g., flu), to less-understood chronic diseases (e.g., diabetes), classical small molecule drugs often possess significant side-effects (a major cause of drug withdrawal from market) due to their low- or non-specific targeting. By contrast, therapeutic peptides, which comprise short sequences from naturally occurring peptides/proteins, commonly demonstrate high target specificity, well-characterized modes-of-action, and low or non-toxicity in vivo. Unfortunately, due to their small size, linear permutation, and lack of tertiary structure, peptidic drugs are easily subject to rapid degradation or loss in vivo through chemical and physical routines, thus resulting in a short half-life and reduced therapeutic efficacy, a major drawback that can reduce therapeutic efficiency. However, recent studies demonstrate that the short half-life of peptidic drugs can be significantly extended by various means, including use of enantiomeric or non-natural amino acids (AAs) (e.g., L-AAs replacement with D-AAs), chemical conjugation [e.g., with polyethylene glycol], and encapsulation (e.g., in exosomes). In this context, we provide an overview of the major in vivo degradation forms of small therapeutic peptides in the plasma and anti-degradation strategies. We also update on the progress of small peptide therapeutics that are either currently in clinical trials or are being successfully used in clinical therapies for patients with non-infectious diseases, such as diabetes, multiple sclerosis, and cancer.
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    Interleukin Receptor Associated Kinase 1 Signaling and Its Association with Cardiovascular Diseases
    Zheng, Youjing; He, Jia-Qiang (IMR Press, 2022-03-09)
    Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) directly interact with intracellular interleukin receptor associated kinase (IRAK) family members to initialize innate immune and inflammatory responses following activation by pathogen-associated or hostderived elements. Although four IRAK family members [IRAK1, 2, 3 (i.e., IRAK-M), and 4] are involved in TLR and IL-1R signaling pathways, IL-1R > IRAK1 signaling appears to be the most studied pathway, with sufficient evidence to support its central role linking the innate immune response to the pathogenesis of various diseases, including cancers, metabolic disorders, and non-infectious immune disorders. However, IRAK1's involvement in cardiovascular diseases was only recently revealed and the detailed mechanism underling the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and heart failure (all non-infectious disorders), remains largely unknown with very limited publications to date. This review aims to summarize the overall roles of the IRAK family, especially IRAK1, in mediating the development of cardiovascular diseases.
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    Keratin Microparticles for Drug and Cell Delivery
    Thompson, Marc Aaron (Virginia Tech, 2019-05-02)
    Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.
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    Keratose Hydrogels Promote Vascular Smooth Muscle Differentiation from c-kit+ Human Cardiac Stem Cells: Underlying Mechanism and Therapeutic Potential
    Ledford, Benjamin (Virginia Tech, 2018-03-23)
    Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI).
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    Large Mammalian Animal Models of Heart Disease
    Camacho, Paula; Fan, Huimin; Liu, Zhongmin; He, Jia-Qiang (MDPI, 2016-10-05)
    Due to the biological complexity of the cardiovascular system, the animal model is an urgent pre-clinical need to advance our knowledge of cardiovascular disease and to explore new drugs to repair the damaged heart. Ideally, a model system should be inexpensive, easily manipulated, reproducible, a biological representative of human disease, and ethically sound. Although a larger animal model is more expensive and difficult to manipulate, its genetic, structural, functional, and even disease similarities to humans make it an ideal model to first consider. This review presents the commonly-used large animals—dog, sheep, pig, and non-human primates—while the less-used other large animals—cows, horses—are excluded. The review attempts to introduce unique points for each species regarding its biological property, degrees of susceptibility to develop certain types of heart diseases, and methodology of induced conditions. For example, dogs barely develop myocardial infarction, while dilated cardiomyopathy is developed quite often. Based on the similarities of each species to the human, the model selection may first consider non-human primates—pig, sheep, then dog—but it also depends on other factors, for example, purposes, funding, ethics, and policy. We hope this review can serve as a basic outline of large animal models for cardiovascular researchers and clinicians.
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    Rho-Associated Kinase Inhibitor (Y-27632) Attenuates Doxorubicin-Induced Apoptosis of Human Cardiac Stem Cells
    Kan, Lijuan; Smith, Aubrie; Chen, Miao; Ledford, Benjamin T.; Fan, Huimin; Liu, Zhongmin; He, Jia-Qiang (PLoS One, 2015-12-08)
    Background Recent clinical trials using c-kit+ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis. Methods and Results c-kit+ CSCs were cultured in CSC medium for 3–5 days followed by 48hr treatment with 0 to 10μM Y-27632 alone, 0 to 1.0μM Dox alone, or Y-27632 followed by Dox (48hrs). Cell viability, toxicity, proliferation, morphology, migration, Caspase-3 activity, expression levels of apoptotic-related key proteins and c-kit+ were examined. Results showed that 48hr treatment with Y-27632 alone did not result in great changes in c-kit+ expression, proliferation, Caspase-3 activity, or apoptosis; however cell viability was significantly increased and cell migration was promoted. These effects likely involve the ROCK/Actin pathways. In contrast, 48hr treatment with Dox alone dramatically increased Caspase-3 activity, resulting in cell death. Although Y-27632 alone did not affect the expression levels of apoptotic-related key factors (p-Akt, Akt, Bcl-2, Bcl-xl, Bax, cleaved Caspase-3, and Caspase-3) under basal conditions, it significantly inhibited the Dox-induced increase in cleaved Caspase-3 and reduced cell death under Dox treatment. Conclusions We conclude that preconditioning human CSCs with Y-27632 significantly reduces Dox-induced cell death and possibly involves the cleaved Caspase-3 and ROCK/Actin pathways. The beneficial effects of Y-27632 may be applied to stem cell-based therapy to increase cell survival rates after transplantation or to act as a cardiac protective agent for Dox-treated cancer patients.
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    Small Therapeutic Peptides: In vitro pharmacokinetics of alpha-carboxyl terminus 11 peptide in rat plasma
    Tasdemiroglu, Yagmur (Virginia Tech, 2021-06-04)
    Cardiovascular diseases affect one third of the U.S. population and are the number one cause of death globally. Acute myocardial infarction is one of the most catastrophic cardiovascular diseases that permanently alters patient's lives. Small molecule drugs, surgery, medical devices and lifestyle changes are the current treatment methods that only address symptoms and fail to cure cardiovascular disorders. Small therapeutic peptides are emerging methods to treat diseases ranging from cancer to auto-immune disorders. Due to their nature, they are non-toxic, non-immunogenic, biocompatible and highly target specific. However, because of their small size and lack of tertiary structure, they have a very short half-life. Alpha-carboxyl terminus 11 peptide (αCT11) is a 9 amino acid long small peptide that has shown to promote left ventricular function recovery when mouse hearts are perfused with the peptide prior to an ischemia-reperfusion injury. This study investigates the in vitro pharmacokinetics of αCT11 in rat plasma in the presence of protease and phosphatase inhibitor cocktails to provide a method to delay its degradation and to understand the degradation pattern of the peptide in vitro. The effect of time, temperature, presence of inhibitors and sex are investigated. Results have shown that while sex does not have a significant effect on αCT11 degradation, time and temperature significantly promote its degradation. Utilization of inhibitors also leads to a pronounced delay in αCT11 degradation, as the amount of αCT11 remaining in plasma increases from almost undetectable to 15-16% upon introduction of inhibitors. These results indicate that αCT11 degradation can be delayed significantly when inhibitor cocktails are used, bringing αCT11 closer to being used in a clinical setting to address ischemia-reperfusion injuries.