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Open Access Publications from the University of California

Scholarly Works (13 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2015)
Cardiovascular disease is the leading cause of mortality in the United States. Myocardial infarction (MI) induces massive cellular death and leads to a decline in cardiac function. Cardiomyocytes have limited proliferative capacity sparking interests in molecular and cellular strategies to promote stem cell conversion into new cardiomyocytes. Cardiac progenitor cells (CPCs) are tissue resident stem cells that give rise to cardiomyogenic structures. Although, CPCs introduced into the heart confer improvements in cardiac function after MI, these effects are not sufficient to support complete heart regeneration and prevent heart failure. Studying molecular pathways that contribute to CPC survival and commitment is essential in advancing CPC based therapeutic approaches. Ca²⁺/Calmodulin-dependent protein kinase II[Delta]B (CaMKII[Delta]B) regulates survival and growth in cardiomyocytes. However, the role of CaMKII[Delta] in CPCs has not been previously explored. CPCs increase nuclear CaMKII after MI and in vitro differentiation suggesting that CaMKII[Delta]B contributes to the regulation of CPC commitment. Overexpression of CaMKII[Delta]B in CPCs reduces proliferation, enhances resistance to death and increases cardiac specific differentiation. CaMKII[Delta]B may serve as a novel modulatory kinase to promote CPC survival and commitment. Despite increasing use of stem cells for regenerative-based cardiac therapy, the optimal stem cell population(s) remains uncertain. In the past decade there has been increasing interest and characterization of stem cell populations reported to directly and/or indirectly contributes to cardiac regeneration through processes of cardiomyogenic commitment and / or release of cardioprotective paracrine factors. Future therapies require development of unprecedented concepts to enhance myocardial healing. Combinatorial cell therapy utilizing CPCs and bone marrow derived mesenchymal stem cells (MSCs) promote enhanced reparative functions in vivo. However, identifying cell specific mechanisms of cardiac repair are difficult using dual cell systems. Here, we performed cell fusion between CPCs and MSCs to obtain hybrids with combined cell characteristics called CardioChimeras. Our ideal cell therapy is to combine the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion
    Cover page: Gene Therapy and Cell Fusion Enhances Stem Cell Mediated Cardiac Repair
    • Thesis
    • Peer Reviewed
    UCLA Electronic Theses and Dissertations (2023)

    Background:Myocardial infarctions (MI) are a leading cause of death worldwide. Unfortunately, the adult heart cannot generate new vessels after MI leading to impaired myocardial perfusion and function. This study aims to identify reprogramming factors during embryonic angiogenesis, which may enhance neovascularization in the adult heart after MI. Our lab discovered 56 novel secreted ligand genes expressed by epicardial cells during development. One factor, collagen, type XVIII, alpha 1 (Col18a1), an angiogenesis inhibitor, has not been thoroughly investigated in the heart.

    Methods:Wilm’s tumor gene 1 (Wt1) transgenic and C57BL/6 mouse strains were used to investigate Col18a1 expression at embryonic (E) and early post-natal (P) time points. Overexpression of COL18A1 in the epicardium was performed at E12.5 to investigate alterations in coronary angiogenesis using gene expression and immunohistochemical analyses.

    Results:Col18a1 increased in Wt1+ epicardial cells during coronary vasculature formation, peaking at E16.5, and stabilized during the early postnatal periods determined by fluorescence in situ hybridization assays. Gene expression data was corroborated following immunohistochemical analysis of endostatin, a proteolytically produced C-terminal fragment of COL18A1 and potent antiangiogenic protein. COL18A1 overexpression in the developing epicardium caused dysregulation of endothelial cell (EC) positioning within the epicardium, observed after immunolabeling for ERG+ cells. Gene expression data confirmed dysregulation of EC differentiation with a downregulation in arterial Nrp1 and upregulation of venous Nr2f2.

    Conclusions:Our data suggest that mRNA and protein levels of Col18a1 and endostatin increased during embryogenesis to limit coronary angiogenesis. Overexpression of COL18A1 inhibited migration and preserved a venous and coronary plexus phenotype in ECs. Furthermore, overexpression of COL18A1 produced immature and proliferative epicardial cells, confirmed by an upregulation in epithelial-to-mesenchymal genes. We theorize that inhibition of epicardial cell differentiation by COL18A1 overexpression drives the dysregulation in EC placement and coronary development.

      Cover page: Regulation of Developmental Angiogenesis by Collagen XVIII alpha 1
      • Thesis
      • Peer Reviewed
      UCLA Electronic Theses and Dissertations (2022)

      Background: Pericytes are abluminal mural cells implicated in angiogenesis, remodeling, stabilization, and maturation of microvasculature in the brain, lungs, kidneys, and retina. These cells have been reported to dissociate from the surrounding microvasculature following an ischemic injury. However, little information is known about cardiac pericyte migration and proliferation following a myocardial infarction (MI).

      Methods: CSPG4CreERT2/+;Rosa26 Ai9tdT/+ mice were used to measure pericyte migration and proliferation following a MI time course. CSPG4CreERT2/+;p53flox/flox mice were used to measure the impacts of knocking out p53 within pericytes under healthy and MI conditions.

      Results: Cardiac pericytes increase in size starting at 7-days post-injury and undergo peak proliferation at 14-days post-injury. Cardiac pericytes reached peak migration at distances >45�m 7-days post-injury. No phenotypic differences were observed between p53 KO and WT pericytes at baseline.

      Conclusions: Increases in pericyte proliferation and migration at later time points could indicate that pericytes contribute to the pro-reparative response in the heart after MI.

      • 1 supplemental ZIP
      Cover page: Characterization of Cardiac Pericyte Proliferation and Function Following a Myocardial Infarction
      • Article
      • Peer Reviewed
      Research Grants Program Office (RGPO) Funded Publications (2017)
      PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.
        Cover page: PIM1-minicircle as a therapeutic treatment for myocardial infarction
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2015)
        Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2018)
          Aging severely limits myocardial repair and regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesized that short telomeres activate p53 and induce autophagy to elicit the age-associated change in CPC fate. We isolated CPCs and compared mouse strains with different telomere lengths for phenotypic characteristics of aging. Wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres exhibits early cardiac aging with cardiac dysfunction, hypertrophy, fibrosis, and senescence, as compared with common lab strains FVB and C57 bearing longer telomeres. CAST CPCs with short telomeres demonstrate altered cell fate as characterized by cell cycle arrest, senescence, basal commitment, and loss of quiescence. Elongation of telomeres using a modified mRNA for telomerase restores youthful properties to CAST CPCs. Short telomeres induce autophagy in CPCs, a catabolic protein degradation process, as evidenced by reduced p62 and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation reverses the cell fate to a more youthful phenotype. Mechanistically, cell fate changes induced by short telomeres are partially p53 dependent, as p53 inhibition rescues senescence and commitment observed in CAST CPCs, coincident with attenuation of autophagy. In conclusion, short telomeres activate p53 and autophagy to tip the equilibrium away from quiescence and proliferation toward differentiation and senescence, leading to exhaustion of CPCs. This study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to prevent senescence of CPCs. Stem Cells 2018;36:868-880.
            Cover page: Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell FateCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2015)
            Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood. Using mice with genetic ablation of calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ), which are resistant to pathological cardiac stress, we show that CaMKIIδ regulates the phosphorylation of histone H3 at serine-10 during pressure overload hypertrophy. H3 S10 phosphorylation is strongly increased in the adult mouse heart in the early phase of cardiac hypertrophy and remains detectable during cardiac decompensation. This response correlates with up-regulation of CaMKIIδ and increased expression of transcriptional drivers of pathological cardiac hypertrophy and of fetal cardiac genes. Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3. Robust H3 phosphorylation is detected in both adult ventricular myocytes and in non-cardiac cells in the stressed myocardium, and these signals are abolished in CaMKIIδ-deficient mice after pressure overload. Mechanistically, fetal cardiac genes are activated by increased recruitment of CaMKIIδ and enhanced H3 phosphorylation at hypertrophic promoter regions, both in mice and in human failing hearts, and this response is blunted in CaMKIIδ-deficient mice under stress. We also document that the chaperone protein 14-3-3 binds phosphorylated H3 in response to stress, allowing proper elongation of fetal cardiac genes by RNA polymerase II (RNAPII), as well as elongation of transcription factors regulating cardiac hypertrophy. These processes are impaired in CaMKIIδ-KO mice after pathological stress. The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy.
              • Article
              • Peer Reviewed
              Research Grants Program Office (RGPO) Funded Publications (2019)

              Rationale

              Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed.

              Objective

              Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes.

              Methods and results

              Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium.

              Conclusions

              Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.
                Cover page: Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI)Creative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                Research Grants Program Office (RGPO) Funded Publications (2016)

                Background

                Myocardial infarction is followed by cardiac dysfunction, cellular death, and ventricular remodeling, including tissue fibrosis. S100A4 protein plays multiple roles in cellular survival, and tissue fibrosis, but the relative role of the S100A4 in the myocardium after myocardial infarction is unknown. This study aims to investigate the role of S100A4 in myocardial remodeling and cardiac function following infarct damage.

                Methods and results

                S100A4 expression is low in the adult myocardium, but significantly increased following myocardial infarction. Deletion of S100A4 increased cardiac damage after myocardial infarction, whereas cardiac myocyte-specific overexpression of S100A4 protected the infarcted myocardium. Decreased cardiac function in S100A4 Knockout mice was accompanied with increased cardiac remodeling, fibrosis, and diminished capillary density in the remote myocardium. Loss of S100A4 caused increased apoptotic cell death both in vitro and in vivo in part mediated by decreased VEGF expression. Conversely, S100A4 overexpression protected cells against apoptosis in vitro and in vivo. Increased pro-survival AKT-signaling explained reduced apoptosis in S100A4 overexpressing cells.

                Conclusion

                S100A4 expression protects cardiac myocytes against myocardial ischemia and is required for stabilization of cardiac function after MI.
                  Cover page: S100A4 protects the myocardium against ischemic stressCreative Commons 'BY' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2023)
                  Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.
                    Cover page: Cardiac pericytes mediate the remodeling response to myocardial infarction
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