Outcomes with guideline-directed medical therapy and cardiac implantable electronic device therapies for patients with heart failure with reduced ejection fraction.

Background: Limited real-world evidence exists for outcomes with contemporary guideline-directed medical therapy (GDMT) or GDMT with implantable cardioverter-defibrillator (ICD)/cardiac resynchronization therapy defibrillator (CRT-D) therapy for patients with heart failure with reduced ejection fraction (HFrEF) and left ventricular ejection fraction (LVEF) ≤35%. Objective: The present study aimed to assess survival associated with GDMT or GDMT with ICD/CRT-D therapy. Methods: This retrospective observational study included real-world de-identified data from January 1, 2016, to December 19, 2023, from 24 U.S. institutions per participating institutional agreements (egnite Database; egnite, Inc.). Patients with a diagnosis of HFrEF and an echocardiographic study documenting LVEF ≤35% were included for analysis. Results: Of 43,591 patients with eligible index event of LVEF ≤35%, prescription history through ≥1 year preindex, and no ICD/CRT-D therapy preindex, mean ± standard deviation age at index was 71.2 ± 13.2 years; 14,805 (34.0%) patients were female. At 24 months, an estimated 99.1% (95% confidence interval [CI] 99.0%-99.2%), 89.9% (95% CI 89.7%-90.1%), 54.8% (95% CI 54.4%-55.2%), and 17.2% (95% CI 16.9%-17.5%), had ≥1, 2, 3, or all 4 GDMT classes prescribed, respectively; an estimated 15.7% (95% CI 15.3%-16.1%) had device placement. Of those without a device, by 24 months, an estimated 45.1% (95% CI 44.4%-45.7%) had a documented LVEF >35%. Counts of GDMT classes prescribed as well as ICD/CRT-D device therapy were associated with lower mortality risk in this population, even after adjustment for patient age, sex, and comorbidities. Conclusion: Both GDMT classes prescribed and device therapy were independently associated with lower mortality risk, even in the presence of more GDMT options for this more contemporary population.

COVID-19 Respiratory Failure: Targeting Inflammation on VV-ECMO Support.

The outbreak of novel coronavirus (SARS-CoV-2) that causes the respiratory illness COVID-19 has led to unprecedented efforts at containment due to its rapid community spread, associated mortality, and lack of immunization and treatment. We herein detail a case of a young patient who suffered life-threatening disease and multiorgan failure. His clinical course involved rapid and profound respiratory decompensation such that he required support with venovenous extracorporeal membrane oxygenation (VV-ECMO). He also demonstrated hyperinflammation (C-reactive protein peak 444.6 mg/L) with severe cytokine elevation (Interleukin-6 peak > 3000 pg/ml). Through treatment targeting hyperinflammation, he recovered from critical COVID-19 respiratory failure and required only 160 hours of VV-ECMO support. He was extubated 4 days after decannulation, had progressive renal recovery, and was discharged to home on hospital day 24. Of note, repeat SARS-CoV-2 test was negative 21 days after his first positive test. We present one of the first successful cases of VV-ECMO support to recovery of COVID-19 respiratory failure in North America.

Nestin-Based Reporter Transgenic Mouse Lines.

Nestin expression marks stem and progenitor cells of the neural lineage. Transgenic mouse lines, originally generated to identify neural stem cells, can also help to identify, track, and isolate stem and progenitor cells in a range of tissues of the ectodermal, endodermal, and mesodermal origin. Here, we describe the generation of transgenic mouse lines expressing fluorescent proteins (FP) under the control of critical regulatory elements of the nestin gene and their use for identifying and analyzing adult stem and progenitor cells in various tissues.

Cardiac Regeneration and Stem Cells.

After decades of believing the heart loses the ability to regenerate soon after birth, numerous studies are now reporting that the adult heart may indeed be capable of regeneration, although the magnitude of new cardiac myocyte formation varies greatly. While this debate has energized the field of cardiac regeneration and led to a dramatic increase in our understanding of cardiac growth and repair, it has left much confusion in the field as to the prospects of regenerating the heart. Studies applying modern techniques of genetic lineage tracing and carbon-14 dating have begun to establish limits on the amount of endogenous regeneration after cardiac injury, but the underlying cellular mechanisms of this regeneration remained unclear. These same studies have also revealed an astonishing capacity for cardiac repair early in life that is largely lost with adult differentiation and maturation. Regardless, this renewed focus on cardiac regeneration as a therapeutic goal holds great promise as a novel strategy to address the leading cause of death in the developed world.

Lack of thrombospondin-2 reduces fibrosis and increases vascularity around cardiac cell grafts.

BACKGROUND: Fibrosis around cardiac cell injections represents an obstacle to graft integration in cell-based cardiac repair. Thrombospondin-2 (TSP-2) is a pro-fibrotic, anti-angiogenic matricellular protein and an attractive target for therapeutic knockdown to improve cardiac graft integration and survival. METHODS: We used a TSP-2 knockout (KO) mouse in conjunction with a fetal murine cardiomyocyte grafting model to evaluate the effects of a lack of TSP-2 on fibrosis, vascular density, and graft size in the heart. RESULTS: Two weeks after grafting in the uninjured heart, fibrosis area was reduced 4.5-fold in TSP-2 KO mice, and the thickness of the peri-graft scar capsule was reduced sevenfold compared to wild-type (WT). Endothelial cell density in the peri-graft region increased 2.5-fold in the absence of TSP-2, and cardiomyocyte graft size increased by 46% in TSP-2 KO hearts. CONCLUSIONS: TSP-2 is a key regulator of fibrosis and angiogenesis following cell grafting in the heart, and its absence promotes better graft integration, vascularization, and survival. SUMMARY: Fibrosis around cardiac cell injections impairs graft integration in cell-based cardiac repair. TSP-2 is a pro-fibrotic, anti-angiogenic matricellular protein. Using a TSP-2-knockout mouse model and cardiac cell transplantation, we found significantly reduced fibrosis and increased endothelial cell density in the peri-graft region. Thus, TSP-2 is an attractive target for therapeutic knockdown to improve cardiac graft integration and survival.

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host­graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

A repair "kit" for the infarcted heart.

Transplanted, c-kit expressing marrow-derived progenitors can enhance the function of an infarcted heart, but the mechanism remains unclear. In this issue of Cell Stem Cell, Loffredo et al. (2011) provide evidence that hematopoietic precursors do not differentiate into new cardiomyocytes but, rather, stimulate production of new cardiomyocytes from endogenous progenitors.

Cardiogenesis from human embryonic stem cells.

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-ß superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/ß-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/ß-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

Neural potential of a stem cell population in the hair follicle.

The bulge region of the hair follicle serves as a repository for epithelial stem cells that can regenerate the follicle in each hair growth cycle and contribute to epidermis regeneration upon injury. Here we describe a population of multipotential stem cells in the hair follicle bulge region; these cells can be identified by fluorescence in transgenic nestin-GFP mice. The morphological features of these cells suggest that they maintain close associations with each other and with the surrounding niche. Upon explantation, these cells can give rise to neurosphere-like structures in vitro. When these cells are permitted to differentiate, they produce several cell types, including cells with neuronal, astrocytic, oligodendrocytic, smooth muscle, adipocytic, and other phenotypes. Furthermore, upon implantation into the developing nervous system of chick, these cells generate neuronal cells in vivo. We used transcriptional profiling to assess the relationship between these cells and embryonic and postnatal neural stem cells and to compare them with other stem cell populations of the bulge. Our results show that nestin-expressing cells in the bulge region of the hair follicle have stem cell-like properties, are multipotent, and can effectively generate cells of neural lineage in vitro and in vivo.

Regulation of the intermediate filament protein nestin at rodent neuromuscular junctions by innervation and activity.

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Expression of nestin-green fluorescent protein transgene marks oval cells in the adult liver.

Oval cells, which become apparent in the liver after chronic injury, serve as bipotent progenitors for differentiated hepatocytes and cholangiocytes. We found that, in the liver of adult transgenic mice in which expression of green fluorescent protein (GFP) is driven by regulatory elements of the nestin gene, the GFP signal marks a subpopulation of small epithelial cells that meet the criteria for oval cells, including morphology, localization, antigenic profile, and reactivity in response to injury. In the regenerating and developing liver, we also found nestin-GFP-positive cells that express hepatocyte markers; such cells may correspond to transiently appearing differentiating progeny of oval cells. During development, GFP-expressing cells in the liver emerge relatively late, after the appearance of differentiated hepatocytes and cholangiocytes. Our results suggest that nestin-GFP cells in the liver correspond to a specialized cell type whose primary function may be to serve as a reserve for adult liver epithelial cell types.

Neural stem and progenitor cells in nestin-GFP transgenic mice.

Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.

Nestin expression in hair follicle sheath progenitor cells.

The intermediate filament protein, nestin, marks progenitor cells of the CNS. Such CNS stem cells are selectively labeled by placing GFP under the control of the nestin regulatory sequences. During early anagen or growth phase of the hair follicle, nestin-expressing cells, marked by GFP fluorescence in nestin-GFP transgenic mice, appear in the permanent upper hair follicle immediately below the sebaceous glands in the follicle bulge. This is where stem cells for the hair follicle outer-root sheath are thought to be located. The relatively small, oval-shaped, nestin-expressing cells in the bulge area surround the hair shaft and are interconnected by short dendrites. The precise locations of the nestin-expressing cells in the hair follicle vary with the hair cycle. During telogen or resting phase and in early anagen, the GFP-positive cells are mainly in the bulge area. However, in mid- and late anagen, the GFP-expressing cells are located in the upper outer-root sheath as well as in the bulge area but not in the hair matrix bulb. These observations show that the nestin-expressing cells form the outer-root sheath. Results of the immunohistochemical staining showed that nestin, GFP, keratin 5/8, and keratin 15 colocalize in the hair follicle bulge cells, outer-root sheath cells, and basal cells of the sebaceous glands. These data indicate that nestin-expressing cells, marked by GFP, in the hair follicle bulge are indeed progenitors of the follicle outer-root sheath. The expression of the unique protein, nestin, in both neural stem cells and hair follicle stem cells suggests their possible relation.