Low-density lipoprotein cholesterol levels and lipid-modifying therapy prescription patterns in the real world: An analysis of more than 33,000 high cardiovascular risk patients in Japan.

BACKGROUND AND AIMS: Low-density lipoprotein cholesterol (LDL-C) is a key modifiable risk factor in the development of cardiovascular (CV) disease. In 2012, the Japan Atherosclerosis Society (JAS) issued guidelines recommending statins as first-line pharmacotherapy for lowering LDL-C in patients at high risk for CV events. This study assessed achievement of recommended LDL-C goals and lipid-modifying therapy (LMT) use in a high CV risk population in Japan. METHODS: Patients from the Medical Data Vision (MDV) database, an electronic hospital-based claims database in Japan, who met the following inclusion criteria were included in this study: LDL-C measurement in 2013; ≥20 years of age; ≥2 years representation in the database; and a high CV risk condition (recent acute coronary syndrome (ACS), other coronary heart disease (CHD), ischemic stroke, peripheral arterial disease (PAD) or diabetes). LDL-C goal attainment was assessed based on LDL-C targets in the JAS guidelines. RESULTS: A total of 33,325 high CV risk patients met the inclusion criteria. Overall, 68% of the cohort achieved guideline recommended LDL-C targets, with only 42% receiving current treatment with statins. Attainment of LDL-C goals was 68% for ACS, 55% for CHD, and 80% each for ischemic stroke, PAD, and diabetes patients. Concomitant use of non-statin LMTs was low. CONCLUSIONS: In a high CV risk population in a routine care setting in Japan, guideline recommended LDL-C goal attainment and utilization of statins and other LMT was low. In addition, physicians appeared to be more likely to consider the initiation of statins in patients with higher baseline LDL-C levels.

Upregulation of leukemia inhibitory factor (LIF) during the early stage of optic nerve regeneration in zebrafish.

Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3-5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

Regeneration-associated genes on optic nerve regeneration in fish retina.

It has been well documented that fish central nervous system, including retina and optic nerve, can regenerate and recover its function after nerve injury. Within a few decades, a number of regeneration-associated genes (RAGs) have been identified in fish retina following optic nerve injury (ONI). RAGs can be classified into two groups: cell survival- and axonal outgrowth-related genes. In fish retina after ONI, cell survival-related genes were upregulated in 1-6 days after ONI, which corresponds to the preparation stage for cell survival and axonal sprouting. Subsequently, axonal outgrowth-related genes were upregulated in 1-6 weeks after ONI, which corresponds to the axonal regrowth stage. Recently, we've found a novel type of RAGs, dedifferentiation-related genes, that are upregulated in overlapping time between cell survival and axonal regrowth (3-10 days after ONI). In this chapter we summarize these three types of RAGs that promote optic nerve regeneration in the fish retina after ONI.

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish.

Unlike mammals, fish motor function can recover within 6-8weeks after spinal cord injury (SCI). The motor function of zebrafish is regulated by dual control; the upper motor neurons of the brainstem and motor neurons of the spinal cord. In this study, we aimed to investigate the framework behind the regeneration of upper motor neurons in adult zebrafish after SCI. In particular, we investigated the cell survival of axotomized upper motor neurons and its molecular machinery in zebrafish brain. As representative nuclei of upper motor neurons, we retrogradely labeled neurons in the nucleus of medial longitudinal fasciculus (NMLF) and the intermediate reticular formation (IMRF) using a tracer injected into the lesion site of the spinal cord. Four to eight neurons in each thin sections of the area of NMLF and IMRF were successfully traced at least 1-15days after SCI. TUNEL staining and BrdU labeling assay revealed that there was no apoptosis or cell proliferation in the axotomized neurons of the brainstem at various time points after SCI. In contrast, axotomized neurons labeled with a neurotracer showed increased expression of anti-apoptotic factors, such as Bcl-2 and phospho-Akt (p-Akt), at 1-6days after SCI. Such a rapid increase of Bcl-2 and p-Akt protein levels after SCI was quantitatively confirmed by western blot analysis. These data strongly indicate that upper motor neurons in the NMLF and IMRF can survive and regrow their axons into the spinal cord through the rapid activation of anti-apoptotic molecules after SCI. The regrowing axons from upper motor neurons reached the lesion site at 10-15days and then crossed at 4-6weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI.

HSP70, the earliest-induced gene in the zebrafish retina during optic nerve regeneration: its role in cell survival.

Fish retinal ganglion cells (RGCs) can survive and regrow their axons after optic nerve injury. Injured RGCs express anti-apoptotic proteins, such as Bcl-2, after nerve injury; however, upstream effectors of this anti-apoptotic protein are not yet fully understood. Heat shock proteins (HSPs) play a crucial role in cell survival against various stress conditions. In this study, we focused on HSP70 expression in the zebrafish retina after optic nerve injury. HSP70 mRNA and protein levels increased rapidly 2.3-fold in RGCs by 1-6 h after injury and returned to control levels by 1-3 days. HSP70 transcription is regulated by heat shock factor 1 (HSF1). HSF1 mRNA and phosphorylated-HSF1 protein rapidly increased by 2.2-fold in RGCs 0.5-6 h after injury. Intraocular injection of HSP inhibitor I significantly suppressed the induction of HSP70 expression after nerve injury. It also suppressed Bcl-2 protein induction and resulted in TUNEL-positive cell death of RGCs at 5 days post-injury. Zebrafish treated with HSP inhibitor I retarded axonal elongation or visual function after injury, as analyzed by GAP43 expression and behavioral analysis of optomotor response, respectively. These results strongly indicate that HSP70, the earliest induced gene in the zebrafish retina after optic nerve injury, is a crucial factor for RGCs survival and optic nerve regeneration in fish.

Growth-associated protein43 (GAP43) is a biochemical marker for the whole period of fish optic nerve regeneration.

In adult visual system, goldfish can regrow their axons and fully restore their visual function even after optic nerve transection. The optic nerve regeneration process in goldfish is very long and it takes about a half year to fully recover visual function via synaptic refinement. Therefore, we investigated time course of growth-associated protein 43 (GAP43) expression in the goldfish retina for over 6 months after axotomy. In the control retina, very weak immunoreactivity could be seen in the retinal ganglion cells (RGCs). The immunoreactivity of GAP43 started to increase in the RGCs at 5 days, peaked at 7-20 days and then gradually decreased at 30-40 days after axotomy. The weak but significant immunoreactivity of GAP43 in the RGCs continued during 50-90 days and slowly returned to the control level by 180 days after lesion. The levels of GAP43 mRNA showed a biphasic pattern; a short-peak increase (9-folds) at 1-3 weeks and a long plateau increase (5-folds) at 50-120 days after axotomy. Thereafter, the levels declined to the control value by 180 days after axotomy. The changes of chasing behavior of pair of goldfish with bilaterally axotomized optic nerve also showed a slow biphasic recovery pattern in time course. Although further experiment is needed to elucidate the role of GAP43 in the regrowing axon terminals, the GAP43 is a good biochemical marker for monitoring the whole period of optic nerve regeneration in fish.

A hypoplastic retinal lamination in the purpurin knock down embryo in zebrafish.

Recently, we cloned a photoreceptor-specific purpurin cDNA from axotomized goldfish retina. In the present study, we investigate the structure of zebrafish purpurin genomic DNA and its function during retinal development. First, we cloned a 3.7-kbp genomic DNA fragment including 1.4-kbp 5'-flanking region and 2.3-kbp full-length coding region. In the 1.4-kbp 5'-upstream region, there were some cone-rod homeobox (crx) protein binding motifs. The vector of the 1.4-kbp 5'-flanking region combined with the reporter GFP gene showed specific expression of this gene only in the photoreceptors. Although the first appearance time of purpurin mRNA expression was a little bit later (40 hpf) than that of crx (17-24 hpf), the appearance site was identical to the ventral part of the retina. Next, we made purpurin or crx knock down embryos with morpholino antisense oligonucleotides. The both morphants (purpurin and crx) showed similar abnormal phenotypes in the eye development; small size of eyeball and lacking of retinal lamination. Furthermore, co-injection of crx morpholino and purpurin mRNA significantly rescued these abnormalities. These data strongly indicate that purpurin is a key molecule for the cell differentiation during early retinal development in zebrafish under transcriptional crx regulation.

Purpurin is a key molecule for cell differentiation during the early development of zebrafish retina.

Recently, we cloned purpurin cDNA as an upregulated gene in the axotomized fish retina. The retina-specific protein was secreted from photoreceptors to ganglion cell layer during an early stage of optic nerve regeneration in zebrafish retina. The purpurin worked as a trigger molecule for axonal regrowth in adult injured fish retina. During zebrafish development, purpurin mRNA first appeared in ventral retina at 2 days post-fertilization (dpf) and spread out to the outer nuclear layer at 3 dpf. Here, we investigated the role of purpurin for zebrafish retinal development using morpholino gene knockdown technique. Injection of purpurin morpholino into the 1-2 cell stage of embryos significantly inhibited the transcriptional and translational expression of purpurin at 3 dpf. In the purpurin morphant, the eyeball was significantly smaller and retinal lamination of nuclear and plexiform layers was not formed at 3 dpf. Retinal cells of purpurin morphants were still proliferative and undifferentiated at 3 dpf. The visual function of purpurin morphant estimated by optomotor response was also suppressed at 5 dpf. By contrast, the control morphants with random sequence morpholino showed retinal lamination with distinct layers and differentiated cells at 3 dpf. These results strongly suggest that purpurin is a key molecule for not only optic nerve regeneration in adult but also cell differentiation during early development in embryo.

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo.

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish (Carassius auratus). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5-40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5-40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo. None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.

Involvement of retinoic acid signaling in goldfish optic nerve regeneration.

Recently, we identified a retina-specific retinol-binding protein, purpurin, as a trigger molecule in the early stage of goldfish optic nerve regeneration. Purpurin protein was secreted by photoreceptors to injured ganglion cells, at 2-5 days after optic nerve injury. Purpurin bound to retinol induced neurite outgrowth in retinal explant cultures and retinoic acid (RA) had a comparable effect on neurite outgrowth. These results indicate that purpurin acts as a retinol transporter and facilitates conversion of retinol to RA. Intracellularly, RA is transported into the nucleus with cellular retinoic acid-binding protein IIb (CRABPIIb) and binds with retinoic acid receptor alpha (RARalpha) as a transcriptional regulator of target genes. Here, we investigated the RA signaling through RA synthesis to RARalpha in the goldfish retina during optic nerve regeneration by RT-PCR. Retinaldehyde dehydrogenase 2 (RALDH2; an RA synthetic enzyme) mRNA was increased by 2.7-fold in the retina at 7-10 days and then gradually decreased until 40 days after nerve injury. In contrast, cytochrome P450 26a1 (CYP26a1; an RA degradative enzyme) mRNA was decreased to less than half in the retina at 5-20 days and then gradually returned to the control level by 40 days after nerve injury. CRABPIIb mRNA was increased by 1.5-fold in the retina at 10 days after axotomy, RARalphaa mRNA was increased by 1.8-fold in the retina at 10 days after axotomy. The cellular changes in the RA signaling molecules after optic nerve injury were almost all located in the ganglion cells, as evaluated by in situ hybridization. The present data described for the first time that RA signaling through RALDH2 and CRABPIIb to RARalpha was serially upregulated in the ganglion cells at 7-10 days just after the purpurin induction. Therefore, we conclude that the triggering action of purpurin on optic nerve regeneration is mediated by RA signaling pathway.

Changes of phospho-growth-associated protein 43 (phospho-GAP43) in the zebrafish retina after optic nerve injury: a long-term observation.

The major model animal of optic nerve regeneration in fish is goldfish. A closely related zebrafish is the most popular model system for genetic and developmental studies of vertebrate central nervous system. A few challenging works of optic nerve regeneration have been done with zebrafish. However, knowledge concerning the long term of optic nerve regeneration apparently lacks in zebrafish. In the present study, therefore, we followed changes of zebrafish behavior and phosphorylated form of growth-associated protein 43 (phospho-GAP43) expression in the zebrafish retina over 100 days after optic nerve transection. Optomotor response was fast recovered by 20-25 days after axotomy whereas chasing behavior (a schooling behavior) was slowly recovered by 80-100 days after axotomy. The temporal pattern of phospho-GAP43 expression showed a biphasic increase, a short-peak (12 folds) at 1-2 weeks and a long-plateau (4 folds) at 1-2 months after axotomy. The recovery of optomotor response well correlated with projection of growing axons to the tectum, whereas the recovery of chasing behavior well correlated with synaptic refinement of retinotectal topography. The present data strongly suggest that phospho-GAP43 plays an active role in both the early and late stages of optic nerve regeneration in fish.

Purpurin expression in the zebrafish retina during early development and after optic nerve lesion in adults.

Purpurin, a retina-specific protein, is known to play a role in cell adhesion during development of the chicken retina. Although purpurin has been significantly detected in adult chicken retina, its function in the matured retina is not well understood. Therefore, to determine the expression pattern of purpurin in the retina, we simultaneously investigated expression patterns of purpurin in the zebrafish retina during development in larvae and optic nerve regeneration after nerve transection in adults. In early development, levels of purpurin suddenly increased in the zebrafish retina 3 to 5 days after fertilization, and purpurin-positive immunoreactivity was diffusely located in all retinal layers. In contrast, levels of purpurin mRNA rapidly increased in the adult retina 1-3 days after optic nerve transection, and rapidly declined by 10 days after injury. Signal for purpurin mRNA was seen only in photoreceptors. Immunohistochemistry showed that levels of purpurin protein were also increased in the retina 1-3 days after nerve injury, but positive staining was located in photoreceptors and ganglion cells, and the staining in ganglion cells was stronger than that in photoreceptors. Thus, the transient expression of purpurin protein was greatly different during development and optic nerve regeneration. In the former, purpurin may be required in all retinal layers, whereas in the latter, purpurin may be required for injured ganglion cells.

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury.

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.

Role of purpurin as a retinol-binding protein in goldfish retina during the early stage of optic nerve regeneration: its priming action on neurite outgrowth.

Unlike mammals, the fish optic nerve can regenerate after injury. So far, many growth or trophic factors have been shown as an axon-regenerating molecule. However, it is totally unknown what substance regulates or triggers the activity of these factors on axonal elongation. Therefore, we constructed a goldfish retina cDNA library prepared from the retina treated with optic nerve transection 5 d previously, when it was just before regrowing optic axons after injury. A cDNA clone for goldfish purpurin for which expression was upregulated during the early stage of optic nerve regeneration was isolated from the retina cDNA library. Purpurin was discovered as a secretory retinol-binding protein in developing chicken retinas. Levels of purpurin mRNA and protein transiently increased and rapidly decreased 2-5 d and 10 d after axotomy, respectively. Purpurin mRNA was localized to the photoreceptor cells, whereas the protein was diffusely found in all of the retinal layers. A recombinant purpurin alone did not affect any change of neurite outgrowth in explant culture of the control retina, whereas a concomitant addition of the recombinant purpurin and retinol first induced a drastic enhancement of neurite outgrowth. Furthermore, the action of retinol-bound purpurin was effective only in the control (untreated) retinas but not in those primed (treated) with a previous optic nerve transection. Thus, purpurin with retinol is the first candidate molecule of priming neurite outgrowth in the early stage of optic nerve regeneration in fish.