Rationale: Fibrosis is mediated partly by extracellular matrix–depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. Objective: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. Methods and Results: We showed that Thy1 + CD45 − CD31 − CD11b − Ter119 − cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. Conclusions: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

BackgroundEffects of extreme sleep duration on risk of mortality and cardiovascular outcomes remain controversial. We aimed to quantify the dose‐response relationships of sleep duration with risk of all‐cause mortality, total cardiovascular disease, coronary heart disease, and stroke.Methods and ResultsPubMed and Embase were systematically searched for prospective cohort studies published before December 1, 2016, that examined the associations between sleep duration and at least 1 of the 4 outcomes in generally healthy populations. U‐shaped associations were indicated between sleep duration and risk of all outcomes, with the lowest risk observed for ≈7‐hour sleep duration per day, which was varied little by sex. For all‐cause mortality, when sleep duration was <7 hours per day, the pooled relative risk (RR) was 1.06 (95% CI, 1.04–1.07) per 1‐hour reduction; when sleep duration was >7 hours per day, the pooled RR was 1.13 (95% CI, 1.11–1.15) per 1‐hour increment. For total cardiovascular disease, the pooled RR was 1.06 (95% CI, 1.03–1.08) per 1‐hour reduction and 1.12 (95% CI, 1.08–1.16) per 1‐hour increment of sleep duration. For coronary heart disease, the pooled RR was 1.07 (95% CI, 1.03–1.12) per 1‐hour reduction and 1.05 (95% CI, 1.00–1.10) per 1‐hour increment of sleep duration. For stroke, the pooled RR was 1.05 (95% CI, 1.01–1.09) per 1‐hour reduction and 1.18 (95% CI, 1.14–1.21) per 1‐hour increment of sleep duration.ConclusionsOur findings indicate that both short and long sleep duration is associated with an increased risk of all‐cause mortality and cardiovascular events.

Keratinocytes are implicated in sensory transduction and can influence nociception, but whether these contribute to chronic pain is not known. In neurons, voltage-gated sodium channels (Na(v)) are involved in neuropathic pain and are activated by depolarization. Since keratinocytes can also show changes in membrane potential, we used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the expression of sodium channels in these cells. Na(v)1.1, Na(v)1.6, and Na(v)1.8 were localized within keratinocytes in rat epidermis. In addition, sodium channels contribute to the release of ATP from rat keratinocytes in response to increased [K(+)](o), implicating sodium channels in keratinocyte ligand release and nociception. To examine whether keratinocytes may contribute to human pain states, we analyzed sodium channel expression in human skin biopsies from subjects with complex regional pain syndrome Type 1 (CRPS) and post-herpetic neuralgia (PHN) using immunohistochemistry. Control skin exhibited immunolabeling for Na(v)1.5, Na(v)1.6 and Na(v)1.7. In contrast, painful skin from CRPS and PHN subjects displayed Na(v)1.1, Na(v)1.2, and Na(v)1.8 immunolabeling, in addition to substantially increased signal for Na(v)1.5, Na(v)1.6, Na(v)1.7. These observations lead us to propose that pathological increases in keratinocyte sodium channel expression may contribute to pain by increasing epidermal ATP release, resulting in excessive activation of P2X receptors on primary sensory axons. Consistent with this hypothesis, animal models of neuropathic pain exhibit increases in subcutaneous ATP release and activity of primary sensory neurons, and peripheral administration of P2X antagonists has been shown to reduce neuropathic pain in humans.

Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.

Both Rb and p130 are required for the recruitment of heterochromatin proteins that mediate silencing of proliferation genes in adult cardiac myocytes.

Many patients with traumatic spinal cord injury (SCI) report pain that persists indefinitely and is resistant to available therapeutic approaches. We recently showed that microglia become activated after experimental SCI and dynamically maintain hyperresponsiveness of spinal cord nociceptive neurons and pain-related behaviors. Mechanisms of signaling between microglia and neurons that help to maintain abnormal pain processing are unknown. In this study, adult male Sprague Dawley rats underwent T9 spinal cord contusion injury. Four weeks after injury when lumbar dorsal horn multireceptive neurons became hyperresponsive and when behavioral nociceptive thresholds to mechanical and thermal stimuli were decreased, we tested the hypothesis that prostaglandin E 2 (PGE 2 ) contributes to signaling between microglia and neurons. Immunohistochemical data showed specific localization of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), an upstream regulator of PGE 2 release, to microglial cells and a neuronal localization of the PGE 2 receptor E-prostanoid 2 (EP2). Enzyme immunoassay analysis showed that PGE 2 release was dependent on microglial activation and ERK1/2 phosphorylation. Pharmacological antagonism of PGE 2 release was achieved with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] and the microglial inhibitor minocycline. Cyclooxygenase-2 expression in microglia was similarly reduced by MEK1/2 inhibition. PD98059 and EP2 receptor blockade with AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) resulted in a decrease in hyperresponsiveness of dorsal horn neurons and partial restoration of behavioral nociceptive thresholds. Selective targeting of dorsal horn microglia with the Mac-1-synapse-associated protein (SAP) immunotoxin resulted in reduced microglia staining, reduction in PGE 2 levels, and reversed pain-related behaviors. On the basis of these observations, we propose a PGE 2 -dependent, ERK1/2-regulated microglia-neuron signaling pathway that mediates the microglial component of pain maintenance after injury to the spinal cord.

Several studies have shown that tRNAs can be enzymatically cleaved to generate distinct classes of tRNA-derived fragments (tRF). Here, we report that tRF/miR-1280, a 17-bp fragment derived from tRNA and pre-miRNA, influences Notch signaling pathways that support the function of cancer stem-like cells (CSC) in colorectal cancer progression. tRF/miR-1280 expression was decreased in human specimens of colorectal cancer. Ectopic expression of tRF/miR-1280 reduced cell proliferation and colony formation, whereas its suppression reversed these effects. Mechanistic investigations implicated the Notch ligand JAG2 as a direct target of tRF/miR-1280 binding through which it reduced tumor formation and metastasis. Notably, tRF/miR-1280-mediated inactivation of Notch signaling suppressed CSC phenotypes, including by direct transcriptional repression of the Gata1/3 and miR-200b genes. These results were consistent with findings of decreased levels of miR-200b and elevated levels of JAG2, Gata1, Gata3, Zeb1, and Suz12 in colorectal cancer tissue specimens. Taken together, our results established that tRF/miR-1280 suppresses colorectal cancer growth and metastasis by repressing Notch signaling pathways that support CSC phenotypes. Furthermore, they provide evidence that functionally active miRNA can be derived from tRNA, offering potential biomarker and therapeutic uses. .

Localized increases in synaptic strength constitute a synaptic basis for learning and memory in the CNS and may also contribute to the maintenance of neuropathic pain after spinal cord injury (SCI) through the de novo formation or elaboration of postsynaptic dendritic structures. To determine whether SCI-induced dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain, we analyzed spine morphometry, localization, and functional influence in dorsal horn (DH) neurons in adult rats 1 month after sham surgery, contusion SCI, and SCI treated with a selective inhibitor of Rac1 activation, NSC23766. After SCI, DH neurons located in lamina IV-V exhibited increased spine density, redistributed spines, and mature spines compared with control neurons, which was associated with enhancement of EPSCs in computer simulations and hyperexcitable responsiveness to innocuous and noxious peripheral stimuli in unit recordings in vivo. SCI animals also exhibited symptoms of tactile allodynia and thermal hyperalgesia. Inhibition of the small GTP-binding protein Rac1 ameliorated post-SCI changes in spine morphology, attenuated injury-induced hyperexcitability of wide-dynamic range neurons, and progressively increased pain thresholds over a 3 d period. This suggests that Rac1 is an important intracellular signaling molecule involved in a spinal dendritic spine pathology associated with chronic neuropathic pain after SCI. Our report provides robust evidence for a novel conceptual bridge between learning and memory on the one hand, and neuropathic pain on the other.