Established and Emerging Nucleic Acid Therapies for Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is a genetic disease that leads to elevated low-density lipoprotein cholesterol levels and risk of coronary heart disease. Current therapeutic options for FH remain relatively limited and only partially effective in both lowering low-density lipoprotein cholesterol and modifying coronary heart disease risk. The unique characteristics of nucleic acid therapies to target the underlying cause of the disease can offer solutions unachievable with conventional medications. DNA- and RNA-based therapeutics have the potential to transform the care of patients with FH. Recent advances are overcoming obstacles to clinical translation of nucleic acid-based medications, including greater stability of the formulations as well as site-specific delivery, making gene-based therapy for FH an alternative approach for treatment of FH.

Cardiac shockwave therapy in addition to coronary bypass surgery improves myocardial function in ischaemic heart failure: the CAST-HF trial.

BACKGROUND AND AIMS: In chronic ischaemic heart failure, revascularisation strategies control symptoms but are less effective in improving left ventricular ejection fraction (LVEF). The aim of this trial is to investigate the safety of cardiac shockwave therapy (SWT) as a novel treatment option and its efficacy in increasing cardiac function by inducing angiogenesis and regeneration in hibernating myocardium. METHODS: In this single-blind, parallel-group, sham-controlled trial (cardiac shockwave therapy for ischemic heart failure, CAST-HF; NCT03859466) patients with LVEF ≤40% requiring surgical revascularisation were enrolled. Patients were randomly assigned to undergo direct cardiac SWT or sham treatment in addition to coronary bypass surgery. The primary efficacy endpoint was the improvement in LVEF measured by cardiac magnetic resonance imaging from baseline to 360 days. RESULTS: Overall, 63 patients were randomized, out of which 30 patients of the SWT group and 28 patients of the Sham group attained 1-year follow-up of the primary endpoint. Greater improvement in LVEF was observed in the SWT group (Δ from baseline to 360 days: SWT 11.3%, SD 8.8; Sham 6.3%, SD 7.4, P = .0146). Secondary endpoints included the 6-minute walking test, where patients randomized in the SWT group showed a greater Δ from baseline to 360 days (127.5 m, SD 110.6) than patients in the Sham group (43.6 m, SD 172.1) (P = .028) and Minnesota Living with Heart Failure Questionnaire score on day 360, which was 11.0 points (SD 19.1) for the SWT group and 17.3 points (SD 15.1) for the Sham group (P = .15). Two patients in the treatment group died for non-device-related reasons. CONCLUSIONS: In conclusion, the CAST-HF trial indicates that direct cardiac SWT, in addition to coronary bypass surgery improves LVEF and physical capacity in patients with ischaemic heart failure.

Premature senescence and cardiovascular disease following cancer treatments: mechanistic insights.

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality, especially among the aging population. The "response-to-injury" model proposed by Dr. Russell Ross in 1999 emphasizes inflammation as a critical factor in atherosclerosis development, with atherosclerotic plaques forming due to endothelial cell (EC) injury, followed by myeloid cell adhesion and invasion into the blood vessel walls. Recent evidence indicates that cancer and its treatments can lead to long-term complications, including CVD. Cellular senescence, a hallmark of aging, is implicated in CVD pathogenesis, particularly in cancer survivors. However, the precise mechanisms linking premature senescence to CVD in cancer survivors remain poorly understood. This article aims to provide mechanistic insights into this association and propose future directions to better comprehend this complex interplay.

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

AIMS: Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. METHODS AND RESULTS: The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. CONCLUSION: The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Possible molecular mechanisms underlying the development of atherosclerosis in cancer survivors.

Cancer survivors undergone treatment face an increased risk of developing atherosclerotic cardiovascular disease (CVD), yet the underlying mechanisms remain elusive. Recent studies have revealed that chemotherapy can drive senescent cancer cells to acquire a proliferative phenotype known as senescence-associated stemness (SAS). These SAS cells exhibit enhanced growth and resistance to cancer treatment, thereby contributing to disease progression. Endothelial cell (EC) senescence has been implicated in atherosclerosis and cancer, including among cancer survivors. Treatment modalities for cancer can induce EC senescence, leading to the development of SAS phenotype and subsequent atherosclerosis in cancer survivors. Consequently, targeting senescent ECs displaying the SAS phenotype hold promise as a therapeutic approach for managing atherosclerotic CVD in this population. This review aims to provide a mechanistic understanding of SAS induction in ECs and its contribution to atherosclerosis among cancer survivors. We delve into the mechanisms underlying EC senescence in response to disturbed flow and ionizing radiation, which play pivotal role in atherosclerosis and cancer. Key pathways, including p90RSK/TERF2IP, TGFßR1/SMAD, and BH4 signaling are explored as potential targets for cancer treatment. By comprehending the similarities and distinctions between different types of senescence and the associated pathways, we can pave the way for targeted interventions aim at enhancing the cardiovascular health of this vulnerable population. The insights gained from this review may facilitate the development of novel therapeutic strategies for managing atherosclerotic CVD in cancer survivors.

Role of angiogenic transdifferentiation in vascular recovery.

Tissue repair requires the orchestration of multiple processes involving a multiplicity of cellular effectors, signaling pathways, and cell-cell communication. The regeneration of the vasculature is a critical process for tissue repair and involves angiogenesis, adult vasculogenesis, and often arteriogenesis, which processes enable recovery of perfusion to deliver oxygen and nutrients to the repair or rebuild of the tissue. Endothelial cells play a major role in angiogenesis, whereas circulating angiogenic cells (primarily of hematopoietic origin) participate in adult vasculogenesis, and monocytes/macrophages have a defining role in the vascular remodeling that is necessary for arteriogenesis. Tissue fibroblasts participate in tissue repair by proliferating and generating the extracellular matrix as the structural scaffold for tissue regeneration. Heretofore, fibroblasts were not generally believed to be involved in vascular regeneration. However, we provide new data indicating that fibroblasts may undergo angiogenic transdifferentiation, to directly expand the microvasculature. Transdifferentiation of fibroblasts to endothelial cells is initiated by inflammatory signaling which increases DNA accessibility and cellular plasticity. In the environment of under-perfused tissue, the activated fibroblasts with increased DNA accessibility can now respond to angiogenic cytokines, which provide the transcriptional direction to induce fibroblasts to become endothelial cells. Periphery artery disease (PAD) involves the dysregulation of vascular repair and inflammation. Understanding the relationship between inflammation, transdifferentiation, and vascular regeneration may lead to a new therapeutic approach to PAD.

Epigenetic landscape reveals MECOM as an endothelial lineage regulator.

A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.

Circulating inflammatory biomarker responses in intervention trials in frail and sarcopenic older adults: A systematic review and meta-analysis.

Consistent with the inflammaging concept, cross-sectional associations have been established between inflammatory biomarkers, frailty and sarcopenia. Less certain is the value of inflammatory markers in monitoring potential anti-inflammatory effects of therapeutic interventions targeted at frailty and sarcopenia. The aims of this systematic review and meta-analysis are to determine if there is a measurable change in inflammatory or immune biomarkers in interventions that improve frailty or sarcopenia and 2. To determine if there are specific inflammatory biomarkers with greater sensitivity to change. In total, 3051 articles were scanned with 16, primarily exercise and nutrition interventions, included in the systematic review and 11 in the meta-analysis. At least one of C reactive protein (CRP), interleukin-6 (IL-6) or tumour necrosis factor alpha (TNF-α) was reduced in 10 of the 16 review studies but only 3/13 studies reported reductions in multiple markers. CRP, IL-6 and TNF-α were individually sensitive to change in 5/11, 3/12 and 5/12 studies respectively. In meta-analyses, there was a positive effect favouring intervention conditions for CRP (SMD = -0.28, p = 0.05) and IL-6 (SMD = -0.28, p = 0.05) but not TNF- α (SMD = -0.12, p = 0.48). There were specific issues with the quality of these studies which were not designed with an inflammatory marker as the primary outcome. In conclusion, interventions that improve frailty and sarcopenia can also reduce CRP, IL-6 and TNF-α but the literature lacks consistency. We are unable to conclude any one marker as being superior to others.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Background: Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Methods: Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Results: Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Summary: Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Radiation therapy induces immunosenescence mediated by p90RSK.

Radiation therapy (RT) to the chest increases the patients' risk of cardiovascular disease (CVD). A complete understanding of the mechanisms by which RT induces CVD could lead to specific preventive, therapeutic approaches. It is becoming evident that both genotoxic chemotherapy agents and radiation induce mitochondrial dysfunction and cellular senescence. Notably, one of the common phenotypes observed in cancer survivors is accelerated senescence, and immunosenescence is closely related to both cancer risk and CVD development. Therefore, suppression of immunosenescence can be an ideal target to prevent cancer treatment-induced CVD. However, the mechanism(s) by which cancer treatments induce immunosenescence are incompletely characterized. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 months after RT from 16 thoracic cancer patients. We characterized human immune cell lineages and markers of senescence, DNA damage response (DDR), efferocytosis, and determinants of clonal hematopoiesis of indeterminant potential (CHIP), using mass cytometry (CyTOF). We found that the frequency of the B cell subtype was decreased after RT. Unsupervised clustering of the CyTOF data identified 138 functional subsets of PBMCs. Compared with baseline, RT increased TBX21 (T-bet) expression in the largest B cell subset of Ki67-/DNMT3a+naïve B cells, and T-bet expression was correlated with phosphorylation of p90RSK expression. CD38 expression was also increased in naïve B cells (CD27-) and CD8+ effector memory CD45RA T cells (TEMRA). In vitro, we found the critical role of p90RSK activation in upregulating (1) CD38+/T-bet+ memory and naïve B, and myeloid cells, (2) senescence-associated ß-gal staining, and (3) mitochondrial reactive oxygen species (ROS) after ionizing radiation (IR). These data suggest the crucial role of p90RSK activation in immunosenescence. The critical role of p90RSK activation in immune cells and T-bet induction in upregulating atherosclerosis formation has been reported. Furthermore, T-bet directly binds to the CD38 promoter region and upregulates CD38 expression. Since both T-bet and CD38 play a significant role in the process of immunosenescence, our data provide a cellular and molecular mechanism that links RT-induced p90RSK activation and the immunosenescence with T-bet and CD38 induction observed in thoracic cancer patients treated by RT and suggests that targeting the p90RSK/T-bet/CD38 pathway could play a role in preventing the radiation-associated CVD and improving cancer prognosis by inhibiting immunosenescence.

Cancer treatment-induced NAD+ depletion in premature senescence and late cardiovascular complications.

Numerous studies have revealed the critical role of premature senescence induced by various cancer treatment modalities in the pathogenesis of aging-related diseases. Senescence-associated secretory phenotype (SASP) can be induced by telomere dysfunction. Telomeric DNA damage response induced by some cancer treatments can persist for months, possibly accounting for long-term sequelae of cancer treatments. Telomeric DNA damage-induced mitochondrial dysfunction and increased reactive oxygen species production are hallmarks of premature senescence. Recently, we reported that the nucleus-mitochondria positive feedback loop formed by p90 ribosomal S6 kinase (p90RSK) and phosphorylation of S496 on ERK5 (a unique member of the mitogen-activated protein kinase family that is not only a kinase but also a transcriptional co-activator) were vital signaling events that played crucial roles in linking mitochondrial dysfunction, nuclear telomere dysfunction, persistent SASP induction, and atherosclerosis. In this review, we will discuss the role of NAD+ depletion in instigating SASP and its downstream signaling and regulatory mechanisms that lead to the premature onset of atherosclerotic cardiovascular diseases in cancer survivors.

Endothelial thioredoxin interacting protein (TXNIP) modulates endothelium-dependent vasorelaxation in hyperglycemia.

Endothelial dysfunction, hallmarked by an imbalance between vasoconstriction and vasorelaxation, is associated with diabetes. Thioredoxin Interacting protein (TXNIP), controlled by an exquisitely glucose sensitive gene, is increasingly recognized for its role in diabetes. However, the role of TXNIP in modulating diabetes-related endothelial dysfunction remains unclear. To elucidate the role of TXNIP, we generated two novel mouse strains; endothelial-specific TXNIP knockout (EKO) and a Tet-O inducible, endothelial-specific TXNIP overexpression (EKI). Hyperglycemia was induced by streptozotocin (STZ) treatment in floxed control (fl/fl) and EKO mice. Doxycycline (DOX) was given to EKI mice to induce endothelial TXNIP overexpression. The ablation of endothelial TXNIP improved glucose tolerance in EKO mice. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in STZ-treated fl/fl mice while this STZ impaired vasorelaxation was attenuated in EKO mice. Hyperglycemia induction of NLRP3 and reductions in Akt and eNOS phosphorylation were also mitigated in EKO mice. Overexpression of endothelial TXNIP did not impair glucose tolerance in DOX-treated EKI mice, however induction of endothelial TXNIP led to impaired vasorelaxation in EKI mice. This was associated with increased NLRP3 and reduced Akt and eNOS activation. In conclusion, deletion of endothelial TXNIP is protective against and overexpression of endothelial TXNIP induces endothelial dysfunction; thus, endothelial TXNIP plays a critical role in modulating endothelial dysfunction.

Senescence-Associated Secretory Phenotype as a Hinge Between Cardiovascular Diseases and Cancer.

Overlapping risks for cancer and cardiovascular diseases (CVD), the two leading causes of mortality worldwide, suggest a shared biology between these diseases. The role of senescence in the development of cancer and CVD has been established. However, its role as the intersection between these diseases remains unclear. Senescence was originally characterized by an irreversible cell cycle arrest after a high number of divisions, namely replicative senescence (RS). However, it is becoming clear that senescence can also be instigated by cellular stress, so-called stress-induced premature senescence (SIPS). Telomere shortening is a hallmark of RS. The contribution of telomere DNA damage and subsequent DNA damage response/repair to SIPS has also been suggested. Although cellular senescence can mediate cell cycle arrest, senescent cells can also remain metabolically active and secrete cytokines, chemokines, growth factors, and reactive oxygen species (ROS), so-called senescence-associated secretory phenotype (SASP). The involvement of SASP in both cancer and CVD has been established. In patients with cancer or CVD, SASP is induced by various stressors including cancer treatments, pro-inflammatory cytokines, and ROS. Therefore, SASP can be the intersection between cancer and CVD. Importantly, the conventional concept of senescence as the mediator of cell cycle arrest has been challenged, as it was recently reported that chemotherapy-induced senescence can reprogram senescent cancer cells to acquire "stemness" (SAS: senescence-associated stemness). SAS allows senescent cancer cells to escape cell cycle arrest with strongly enhanced clonogenic growth capacity. SAS supports senescent cells to promote both cancer and CVD, particularly in highly stressful conditions such as cancer treatments, myocardial infarction, and heart failure. As therapeutic advances have increased overlapping risk factors for cancer and CVD, to further understand their interaction may provide better prevention, earlier detection, and safer treatment. Thus, it is critical to study the mechanisms by which these senescence pathways (SAS/SASP) are induced and regulated in both cancer and CVD.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

Induced endothelial cells from peripheral arterial disease patients and neonatal fibroblasts have comparable angiogenic properties.

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.

Fli1+ cells transcriptional analysis reveals an Lmo2-Prdm16 axis in angiogenesis.

A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish Tg(fli1:EGFP)y1 , we identified two endothelial cell populations, high-fli1+ and low-fli1+, by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-fli1 expressing cells (fli1-) with these two (fli1+) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with fli1- and low-fli1+ cells, high-fli1+ cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (prdm16). Prdm16 knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-fli1+ cells at the expense of high-fli1+ cells. In addition, PRDM16 KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (lmo2) also showed reduced prdm16 gene expression combined with impaired angiogenesis. Prdm16 expression was reduced further in endothelial (CD31+) cells compared with CD31- cells isolated from lmo2-mutants (lmo2-mut) embryos. Chromatin immunoprecipitation-PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of prdm16 This work unveils a mechanism by which prdm16 expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair.

New Directions in Therapeutic Angiogenesis and Arteriogenesis in Peripheral Arterial Disease.

The prevalence of peripheral arterial disease (PAD) in the United States exceeds 10 million people, and PAD is a significant cause of morbidity and mortality across the globe. PAD is typically caused by atherosclerotic obstructions in the large arteries to the leg(s). The most common clinical consequences of PAD include pain on walking (claudication), impaired functional capacity, pain at rest, and loss of tissue integrity in the distal limbs that may lead to lower extremity amputation. Patients with PAD also have higher than expected rates of myocardial infarction, stroke, and cardiovascular death. Despite advances in surgical and endovascular procedures, revascularization procedures may be suboptimal in relieving symptoms, and some patients with PAD cannot be treated because of comorbid conditions. In some cases, relieving obstructive disease in the large conduit arteries does not assure complete limb salvage because of severe microvascular disease. Despite several decades of investigational efforts, medical therapies to improve perfusion to the distal limb are of limited benefit. Whereas recent studies of anticoagulant (eg, rivaroxaban) and intensive lipid lowering (such as PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitors) have reduced major cardiovascular and limb events in PAD populations, chronic ischemia of the limb remains largely resistant to medical therapy. Experimental approaches to improve limb outcomes have included the administration of angiogenic cytokines (either as recombinant protein or as gene therapy) as well as cell therapy. Although early angiogenesis and cell therapy studies were promising, these studies lacked sufficient control groups and larger randomized clinical trials have yet to achieve significant benefit. This review will focus on what has been learned to advance medical revascularization for PAD and how that information might lead to novel approaches for therapeutic angiogenesis and arteriogenesis for PAD.

mRNA-Enhanced Cell Therapy and Cardiovascular Regeneration.

mRNA has emerged as an important biomolecule in the global call for the development of therapies during the COVID-19 pandemic. Synthetic in vitro-transcribed (IVT) mRNA can be engineered to mimic naturally occurring mRNA and can be used as a tool to target "undruggable" diseases. Recent advancement in the field of RNA therapeutics have addressed the challenges inherent to this drug molecule and this approach is now being applied to several therapeutic modalities, from cancer immunotherapy to vaccine development. In this review, we discussed the use of mRNA for stem cell generation or enhancement for the purpose of cardiovascular regeneration.

Broad genic repression domains signify enhanced silencing of oncogenes.

Cancers result from a set of genetic and epigenetic alterations. Most known oncogenes were identified by gain-of-function mutations in cancer, yet little is known about their epigenetic features. Through integrative analysis of 11,596 epigenomic profiles and mutations from >8200 tumor-normal pairs, we discover broad genic repression domains (BGRD) on chromatin as an epigenetic signature for oncogenes. A BGRD is a widespread enrichment domain of the repressive histone modification H3K27me3 and is further enriched with multiple other repressive marks including H3K9me3, H3K9me2, and H3K27me2. Further, BGRD displays widespread enrichment of repressed cis-regulatory elements. Shortening of BGRDs is linked to derepression of transcription. BGRDs at oncogenes tend to be conserved across normal cell types. Putative tumor-promoting genes and lncRNAs defined using BGRDs are experimentally verified as required for cancer phenotypes. Therefore, BGRDs play key roles in epigenetic regulation of cancer and provide a direction for mutation-independent discovery of oncogenes.