MicroRNA-150 (miR-150) and Diabetic Retinopathy: Is miR-150 Only a Biomarker or Does It Contribute to Disease Progression?

Diabetic retinopathy (DR) is a chronic disease associated with diabetes mellitus and is a leading cause of visual impairment among the working population in the US. Clinically, DR has been diagnosed and treated as a vascular complication, but it adversely impacts both neural retina and retinal vasculature. Degeneration of retinal neurons and microvasculature manifests in the diabetic retina and early stages of DR. Retinal photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. Chronic inflammation is a hallmark of diabetes and a contributor to cell apoptosis, and retinal photoreceptors are a major source of intraocular inflammation that contributes to vascular abnormalities in diabetes. As the levels of microRNAs (miRs) are changed in the plasma and vitreous of diabetic patients, miRs have been suggested as biomarkers to determine the progression of diabetic ocular diseases, including DR. However, few miRs have been thoroughly investigated as contributors to the pathogenesis of DR. Among these miRs, miR-150 is downregulated in diabetic patients and is an endogenous suppressor of inflammation, apoptosis, and pathological angiogenesis. In this review, how miR-150 and its downstream targets contribute to diabetes-associated retinal degeneration and pathological angiogenesis in DR are discussed. Currently, there is no effective treatment to stop or reverse diabetes-caused neural and vascular degeneration in the retina. Understanding the molecular mechanism of the pathogenesis of DR may shed light for the future development of more effective treatments for DR and other diabetes-associated ocular diseases.

MicroRNA-150 and its target ETS-domain transcription factor 1 contribute to inflammation in diabetic photoreceptors.

Obesity-associated type 2 diabetes (T2D) is on the rise in the United States due to the obesity epidemic, and 60% of T2D patients develop diabetic retinopathy (DR) in their lifetime. Chronic inflammation is a hallmark of obesity and T2D and a well-accepted major contributor to DR, and retinal photoreceptors are a major source of intraocular inflammation and directly contribute to vascular abnormalities in diabetes. However, how diabetic insults cause photoreceptor inflammation is not well known. In this study, we used a high-fat diet (HFD)-induced T2D mouse model and cultured photoreceptors treated with palmitic acid (PA) to decipher major players that mediate high-fat-induced photoreceptor inflammation. We found that PA-elicited microRNA-150 (miR-150) decreases with a consistent upregulation of ETS-domain transcription factor 1 (Elk1), a downstream target of miR-150, in PA-elicited photoreceptor inflammation. We compared wild-type (WT) and miR-150 null (miR-150-/- ) mice fed with an HFD and found that deletion of miR-150 exacerbated HFD-induced photoreceptor inflammation in conjunction with upregulated ELK1. We further delineated the critical cellular localization of phosphorylated ELK1 at serine 383 (pELK1S383 ) and found that decreased miR-150 exacerbated the T2D-induced inflammation in photoreceptors by upregulating ELK1 and pELK1S383 , and knockdown of ELK1 alleviated PA-elicited photoreceptor inflammation.

Circadian regulation in the retina: From molecules to network.

The mammalian retina is the most unique tissue among those that display robust circadian/diurnal oscillations. The retina is not only a light sensing tissue that relays light information to the brain, it has its own circadian "system" independent from any influence from other circadian oscillators. While all retinal cells and retinal pigment epithelium (RPE) possess circadian oscillators, these oscillators integrate by means of neural synapses, electrical coupling (gap junctions), and released neurochemicals (such as dopamine, melatonin, adenosine, and ATP), so the whole retina functions as an integrated circadian system. Dysregulation of retinal clocks not only causes retinal or ocular diseases, it also impacts the circadian rhythm of the whole body, as the light information transmitted from the retina entrains the brain clock that governs the body circadian rhythms. In this review, how circadian oscillations in various retinal cells are integrated, and how retinal diseases affect daily rhythms.

Peptide Lv augments L-type voltage-gated calcium channels through vascular endothelial growth factor receptor 2 (VEGFR2) signaling.

We previously identified peptide Lv, a novel bioactive peptide that enhances the activity of L-type voltage-gated calcium channels (L-VGCCs) in cone photoreceptors. In this study, we verified that peptide Lv was able to augment L-VGCC currents in cardiomyocytes, as well as promote proliferation of endothelial cells. We used a proteomics approach to determine the specific receptors and binding partners of peptide Lv and found that vascular endothelial growth factor receptor 2 (VEGFR2) interacted with peptide Lv. Peptide Lv treatment in embryonic cardiomyocytes stimulated tyrosine autophosphorylation of VEGFR2 and activated its downstream signaling. Peptide Lv activity was blocked by DMH4, a VEGFR2 specific blocker, but not by SCH202676, an allosteric inhibitor of G protein-coupled receptors, suggesting that the activity of peptide Lv was mediated through VEGFR2 signaling. Inhibition of VEGFR tyrosine kinase or its downstream signaling molecules abolished the augmentation of L-VGCCs elicited by peptide Lv in cardiomyocytes. In addition, peptide Lv promoted cell proliferation of cultured human endothelial cells. Calcium entry through L-VGCCs is essential for excitation-contraction coupling in cardiomyocytes. Since peptide Lv was able to augment L-VGCCs through activation of VEGF signaling in cardiomyocytes and promote proliferation of endothelial cells, peptide Lv may play an important role in regulating the cardiovascular system.

A new role for AMP-activated protein kinase in the circadian regulation of L-type voltage-gated calcium channels in late-stage embryonic retinal photoreceptors.

AMP-activated protein kinase (AMPK) is a cellular energy sensor, which is activated when the intracellular ATP production decreases. The activities of AMPK display circadian rhythms in various organs and tissues, indicating that AMPK is involved in the circadian regulation of cellular metabolism. In vertebrate retina, the circadian clocks regulate many aspects of retinal function and physiology, including light/dark adaption, but whether and how AMPK was involved in the retinal circadian rhythm was not known. We hypothesized that the activation of AMPK (measured as phosphorylated AMPK) in the retina was under circadian control, and AMPK might interact with other intracellular signaling molecules to regulate photoreceptor physiology. We combined ATP assays, western blots, immunostaining, patch-clamp recordings, and pharmacological treatments to decipher the role of AMPK in the circadian regulation of photoreceptor physiology. We found that the overall retinal ATP content displayed a diurnal rhythm that peaked at early night, which was nearly anti-phase to the diurnal and circadian rhythms of AMPK phosphorylation. AMPK was also involved in the circadian phase-dependent regulation of photoreceptor L-type voltage-gated calcium channels (L-VGCCs), the ion channel essential for sustained neurotransmitter release. The activation of AMPK dampened the L-VGCC currents at night with a corresponding decrease in protein expression of the L-VGCCα1 pore-forming subunit, while inhibition of AMPK increased the L-VGCC current during the day. AMPK appeared to be upstream of extracellular-signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Hence, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology. We found that in chicken embryonic retina, the activation of AMP-activated protein kinase (AMPK) is under circadian control and anti-phase to the retinal ATP rhythm. While ATP content is higher at night, phosphorylated AMPK (pAMPK) is higher during the day. AMPK appears to be upstream of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and mammalian target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Therefore, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology.

Circadian phase-dependent effect of nitric oxide on L-type voltage-gated calcium channels in avian cone photoreceptors.

Nitric oxide (NO) plays an important role in phase-shifting of circadian neuronal activities in the suprachiasmatic nucleus and circadian behavior activity rhythms. In the retina, NO production is increased in a light-dependent manner. While endogenous circadian oscillators in retinal photoreceptors regulate their physiological states, it is not clear whether NO also participates in the circadian regulation of photoreceptors. In this study, we demonstrate that NO is involved in the circadian phase-dependent regulation of L-type voltage-gated calcium channels (L-VGCCs). In chick cone photoreceptors, the L-VGCCα1 subunit expression and the maximal L-VGCC currents are higher at night, and both Ras-mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (Erk) and Ras-phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) are part of the circadian output pathways regulating L-VGCCs. The NO-cGMP-protein kinase G (PKG) pathway decreases L-VGCCα1 subunit expression and L-VGCC currents at night, but not during the day, and exogenous NO donor or cGMP decreases the phosphorylation of Erk and Akt at night. The protein expression of neural NO synthase (nNOS) is also under circadian control, with both nNOS and NO production being higher during the day. Taken together, NO/cGMP/PKG signaling is involved as part of the circadian output pathway to regulate L-VGCCs in cone photoreceptors. In cone photoreceptors, the protein expression of neural nitric oxide synthase (nNOS) and NO production are under circadian control. NO-cGMP-protein kinase G (PKG) signaling serves in the circadian output pathway to regulate the circadian rhythms of L-type voltage-gated calcium channels (L-VGCCs) in part through regulating the phosphorylation states of extracellular-signal-regulated kinase (Erk) and protein kinase B (Akt).

Peptide Lv Promotes Trafficking and Membrane Insertion of KCa3.1 through the MEK1-ERK and PI3K-Akt Signaling Pathways.

Peptide Lv is a small endogenous secretory peptide that is proangiogenic through hyperpolarizing vascular endothelial cells (ECs) by enhancing the current densities of KCa3.1 channels. However, it is unclear how peptide Lv enhances these currents. One way to enhance the current densities of ion channels is to promote its trafficking and insertion into the plasma membrane. We hypothesized that peptide Lv-elicited KCa3.1 augmentation occurs through activating the mitogen-activated protein kinase kinase 1 (MEK1)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways, which are known to mediate ion channel trafficking and membrane insertion in neurons. To test this hypothesis, we employed patch-clamp electrophysiological recordings and cell-surface biotinylation assays on ECs treated with peptide Lv and pharmaceutical inhibitors of ERK and Akt. Blocking ERK or Akt activation diminished peptide Lv-elicited EC hyperpolarization and increase in KCa3.1 current densities. Blocking PI3K or Akt activation decreased the level of plasma membrane-bound, but not the total amount of KCa3.1 protein in ECs. Therefore, the peptide Lv-elicited EC hyperpolarization and KCa3.1 augmentation occurred in part through channel trafficking and insertion mediated by MEK1-ERK and PI3K-Akt activation. These results demonstrate the molecular mechanisms of how peptide Lv promotes EC-mediated angiogenesis.

Calcineurin serves in the circadian output pathway to regulate the daily rhythm of L-type voltage-gated calcium channels in the retina.

The L-type voltage-gated calcium channels (L-VGCCs) in avian retinal cone photoreceptors are under circadian control, in which the protein expression of the α1 subunits and the current density are greater at night than during the day. Both Ras-mitogen-activated protein kinase (MAPK) and Ras-phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathways are part of the circadian output that regulate the L-VGCC rhythm, while cAMP-dependent signaling is further upstream of Ras to regulate the circadian outputs in photoreceptors. However, there are missing links between cAMP-dependent signaling and Ras in the circadian output regulation of L-VGCCs. In this study, we report that calcineurin, a Ca2+/calmodulin-dependent serine (ser)/threonine (thr) phosphatase, participates in the circadian output pathway to regulate L-VGCCs through modulating both Ras-MAPK and Ras-PI3K-AKT signaling. The activity of calcineurin, but not its protein expression, was under circadian regulation. Application of a calcineurin inhibitor, FK-506 or cyclosporine A, reduced the L-VGCC current density at night with a corresponding decrease in L-VGCCα1D protein expression, but the circadian rhythm of L-VGCCα1D mRNA levels were not affected. Inhibition of calcineurin further reduced the phosphorylation of ERK and AKT (at thr 308) and inhibited the activation of Ras, but inhibitors of MAPK or PI3K signaling did not affect the circadian rhythm of calcineurin activity. However, inhibition of adenylate cyclase significantly dampened the circadian rhythm of calcineurin activity. These results suggest that calcineurin is upstream of MAPK and PI3K-AKT but downstream of cAMP in the circadian regulation of L-VGCCs.

Peptide Lv augments intermediate-conductance calcium-dependent potassium channels (KCa3.1) in endothelial cells to promote angiogenesis.

Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.

Decreased MicroRNA-150 Exacerbates Neuronal Apoptosis in the Diabetic Retina.

Diabetic retinopathy (DR) is a chronic complication associated with diabetes and the number one cause of blindness in working adults in the US. More than 90% of diabetic patients have obesity-associated type 2 diabetes (T2D), and 60% of T2D patients will develop DR. Photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. However, how diabetic insults cause photoreceptor apoptosis remains unclear. In this study, obesity-associated T2D mice and cultured photoreceptors were used to investigate how decreased microRNA-150 (miR-150) and its downstream target were involved in photoreceptor apoptosis. In the T2D retina, miR-150 was decreased with its target ETS-domain transcription factor (ELK1) and phosphorylated ELK1 at threonine 417 (pELK1T417) upregulated. In cultured photoreceptors, treatments with palmitic acid (PA), to mimic a high-fat environment, decreased miR-150 but upregulated ELK1, pELK1T417, and the translocation of pELK1T417 from the cytoplasm to the cell nucleus. Deletion of miR-150 (miR-150-/-) exacerbates T2D- or PA-induced photoreceptor apoptosis. Blocking the expression of ELK1 with small interfering RNA (siRNA) for Elk1 did not rescue PA-induced photoreceptor apoptosis. Translocation of pELK1T417 from cytoplasm-to-nucleus appears to be the key step of diabetic insult-elicited photoreceptor apoptosis.

Rhythmic expression of microRNA-26a regulates the L-type voltage-gated calcium channel alpha1C subunit in chicken cone photoreceptors.

MicroRNAs (miRNAs) modulate gene expression by degrading or inhibiting translation of messenger RNAs (mRNAs). Here, we demonstrated that chicken microRNA-26a (gga-mir-26a) is a key posttranscriptional regulator of photoreceptor L-type voltage-gated calcium channel alpha1C subunit (L-VGCCalpha1C) expression, and its own expression has a diurnal rhythm, thereby explaining the rhythmic nature of L-VGCCalpha1Cs. Circadian oscillators in retinal photoreceptors provide a mechanism that allows photoreceptors to anticipate daily illumination changes. In photoreceptors, L-VGCC activities are under circadian control, which are higher at night and lower during the day. Interestingly, the mRNA level of VGCCalpha1D oscillates, but those for VGCCalpha1C do not. However, the protein expression of both VGCCalpha1C and alpha1D are higher at night in cone photoreceptors. The underlying mechanism regulating L-VGCCalpha1C protein expression was not clear until now. In vitro targeting reporter assays verified that gga-mir-26a specifically targeted the L-VGCCalpha1C 3'-untranslated region, and gga-mir-26a expression in the retina peaked during the day. After transfection with gga-mir-26a, L-VGCCalpha1C protein expression and L-VGCC current density decreased. Therefore, the rhythmic expression of gga-mir-26a regulated the protein expression of the L-VGCCalpha1C subunit. Additionally, both CLOCK (circadian locomoter output cycles kaput) and CREB (cAMP-response element-binding protein-1) activated gga-mir-26a expression in vitro. This result implies that gga-mir-26a might be a downstream target of circadian oscillators. Our work has uncovered new functional roles for miRNAs in the regulation of circadian rhythms in cone photoreceptors. Circadian regulated miRNAs could serve as the link between the core oscillator and output signaling that further govern biological functions.

Decreased miR-150 in obesity-associated type 2 diabetic mice increases intraocular inflammation and exacerbates retinal dysfunction.

INTRODUCTION: Diabetic retinopathy (DR) is the leading cause of blindness among the working population in the USA. Current therapies, including anti-vascular endothelial growth factor treatments, cannot completely reverse the visual defects induced by DR. MicroRNA-150 (miR-150) is a regulator that suppresses inflammation and pathological angiogenesis. In patients with diabetes, miR-150 is downregulated. As chronic inflammation is a major contributor to the pathogenesis of DR, whether diabetes-associated decrease of miR-150 is merely associated with the disease progression or decreased miR-150 causes retinal inflammation and pathological angiogenesis is still unknown. RESEARCH DESIGN AND METHODS: We used high-fat diet (HFD)-induced type 2 diabetes (T2D) in wild type (WT) and miR-150 knockout (miR-150-/-) mice for this study and compared retinal function and microvasculature morphology. RESULTS: We found that WT mice fed with an HFD for only 1 month had a significant decrease of miR-150 in the blood and retina, and retinal light sensitivity also decreased. The miR-150-/- mice on the HFD developed diabetes similar to that of the WT. At 7-8 months old, miR-150-/- mice under normal diet had increased degeneration of retinal capillaries compared with WT mice, indicating that miR-150 is important in maintaining the structural integrity of retinal microvasculature. Deletion of miR-150 worsened HFD-induced retinal dysfunction as early as 1 month after the diet regimen, and it exacerbated HFD-induced T2DR by further increasing retinal inflammation and microvascular degeneration. CONCLUSION: These data suggest that decreased miR-150 caused by obesity or diabetic insults is not merely correlated to the disease progression, but it contributes to the retinal dysfunction and inflammation, as well as the development of DR.

Circadian regulation of ion channels and their functions.

Ion channels are the gatekeepers to neuronal excitability. Retinal neurons of vertebrates and invertebrates, neurons of the suprachiasmatic nucleus (SCN) of vertebrates, and pinealocytes of non-mammalian vertebrates display daily rhythms in their activities. The interlocking transcription-translation feedback loops with specific post-translational modulations within individual cells form the molecular clock, the basic mechanism that maintains the autonomic approximately 24-h rhythm. The molecular clock regulates downstream output signaling pathways that further modulate activities of various ion channels. Ultimately, it is the circadian regulation of ion channel properties that govern excitability and behavior output of these neurons. In this review, we focus on the recent development of research in circadian neurobiology mainly from 1980 forward. We will emphasize the circadian regulation of various ion channels, including cGMP-gated cation channels, various voltage-gated calcium and potassium channels, Na(+)/K(+)-ATPase, and a long-opening cation channel. The cellular mechanisms underlying the circadian regulation of these ion channels and their functions in various tissues and organisms will also be discussed. Despite the magnitude of chronobiological studies in recent years, the circadian regulation of ion channels still remains largely unexplored. Through more investigation and understanding of the circadian regulation of ion channels, the future development of therapeutic strategies for the treatment of sleep disorders, cardiovascular diseases, and other illnesses linked to circadian misalignment will benefit.

Phosphatidylinositol 3 kinase-Akt signaling serves as a circadian output in the retina.

The daily rhythm of L-type voltage-gated calcium channels (L-VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L-VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor-induced ion channel trafficking and membrane insertion. Here, we report that PI3K-Akt signaling participates in the circadian phase-dependent modulation of L-VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L-VGCC current amplitudes and the expression of membrane-bound L-VGCCalpha1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L-VGCCalpha1D subunit compared with non-transfected photoreceptors. Interestingly, both PI3K-Akt and extracellular signal-related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L-VGCC rhythms. Inhibition of either pathway abolished the L-VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L-VGCC rhythms in retina photoreceptors.

Circadian controls outweigh acute illumination effects on the activity of extracellular signal-regulated kinase (ERK) in the retina.

Extracellular signal-regulated kinase (ERK) participates in numerous cellular functions including circadian-related activities. In the retina, the activity of ERK is under circadian control. However, it is not clear whether acute illumination changes or the circadian clocks in the retina have a larger impact on ERK activity, and the cellular distribution of activated ERK (pERK) as a function of circadian time in cone photoreceptors is not known. Chick embryos were exposed to the light or dark for various lengths of time after 12:12h light-dark (LD) cycles, or on the second day of constant darkness after LD entrainment. Retinas were excised after various exposure times and relative ERK activity was determined by western immunoblotting. We also performed immunohistochemical and immunocytochemical stainings on circadian entrained retina sections and dissociated retina cells. There is about a fourfold difference in ERK activity between retinas harvested at circadian time (CT) 4 and CT 16, and the internal circadian control of ERK activity in the retina overcomes external light exposure. Also, during the subjective night, pERK was more apparent in the outer segment of cones, while pERK distribution was more uniform throughout the photoreceptors during the subjective day. Our results imply that the activity of retinal ERK is influenced more by circadian oscillators than acute illumination changes. Hence, the circadian oscillators in retina photoreceptors play a major role in the regulation of photoreceptor physiology, which leads to the circadian control of light sensitivity in photoreceptors.

Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina.

Mitochondrial fission and fusion are dependent on cellular nutritional states, and maintaining this dynamics is critical for the health of cells. Starvation triggers mitochondrial fusion to maintain bioenergetic efficiency, but during nutrient overloads (as with hyperglycemic conditions), fragmenting mitochondria is a way to store nutrients to avoid waste of energy. In addition to ATP production, mitochondria play an important role in buffering intracellular calcium (Ca2+). We found that in cultured 661W cells, a photoreceptor-derived cell line, hyperglycemic conditions triggered an increase of the expression of dynamin-related protein 1 (DRP1), a protein marker of mitochondrial fission, and a decrease of mitofusin 2 (MFN2), a protein for mitochondrial fusion. Further, these hyperglycemic cells also had decreased mitochondrial Ca2+ but increased cytosolic Ca2+. Treating these hyperglycemic cells with melatonin, a multifaceted antioxidant, averted hyperglycemia-altered mitochondrial fission-and-fusion dynamics and mitochondrial Ca2+ levels. To mimic how people most commonly take melatonin supplements, we gave melatonin to streptozotocin- (STZ-) induced type 1 diabetic mice by daily oral gavage and determined the effects of melatonin on diabetic eyes. We found that melatonin was not able to reverse the STZ-induced systemic hyperglycemic condition, but it prevented STZ-induced damage to the neural retina and retinal microvasculature. The beneficial effects of melatonin in the neural retina in part were through alleviating STZ-caused changes in mitochondrial dynamics and Ca2+ buffering.

Newly Identified Peptide, Peptide Lv, Promotes Pathological Angiogenesis.

Background We recently discovered a small endogenous peptide, peptide Lv, with the ability to activate vascular endothelial growth factor receptor 2 and its downstream signaling. As vascular endothelial growth factor through vascular endothelial growth factor receptor 2 contributes to normal development, vasodilation, angiogenesis, and pathogenesis of various diseases, we investigated the role of peptide Lv in vasodilation and developmental and pathological angiogenesis in this study. Methods and Results The endothelial cell proliferation, migration, and 3-dimensional sprouting assays were used to test the abilities of peptide Lv in angiogenesis in vitro. The chick chorioallantoic membranes and early postnatal mice were used to examine its impact on developmental angiogenesis. The oxygen-induced retinopathy and laser-induced choroidal neovascularization mouse models were used for in vivo pathological angiogenesis. The isolated porcine retinal and coronary arterioles were used for vasodilation assays. Peptide Lv elicited angiogenesis in vitro and in vivo. Peptide Lv and vascular endothelial growth factor acted synergistically in promoting endothelial cell proliferation. Peptide Lv-elicited vasodilation was not completely dependent on nitric oxide, indicating that peptide Lv had vascular endothelial growth factor receptor 2/nitric oxide-independent targets. An antibody against peptide Lv, anti-Lv, dampened vascular endothelial growth factor-elicited endothelial proliferation and laser-induced vascular leakage and choroidal neovascularization. While the pathological angiogenesis in mouse eyes with oxygen-induced retinopathy was enhanced by exogenous peptide Lv, anti-Lv dampened this process. Furthermore, deletion of peptide Lv in mice significantly decreased pathological neovascularization compared with their wild-type littermates. Conclusions These results demonstrate that peptide Lv plays a significant role in pathological angiogenesis but may be less critical during development. Peptide Lv is involved in pathological angiogenesis through vascular endothelial growth factor receptor 2-dependent and -independent pathways. As anti-Lv dampened the pathological angiogenesis in the eye, anti-Lv may have a therapeutic potential to treat pathological angiogenesis.

Somatostatin peptides produce multiple effects on gating properties of native cone photoreceptor cGMP-gated channels that depend on circadian phase and previous illumination.

A subpopulation of avian amacrine cells expresses somatostatin-14 (SS14) and somatostatin-28 (SS28), which provide a potential efferent limb for light-dependent regulation of photoreceptors. Here, we demonstrate that SS14 and SS28 modulate cone photoreceptor cGMP-gated channels (CNGCs) through multiple mechanisms. In chicken cones cultured in constant darkness for 2 d after previous entrainment to light-dark (LD) cycles or in cells maintained in LD, application of 100 nm SS14 or 100 nm SS28 for either 15 min or 2 h caused a decrease in the sensitivity of CNGCs to cGMP during the night, at circadian time 16 (CT16)-CT20 or zeitgeber time 16 (ZT16)-ZT20. SS14 had no effect during the day (CT4-CT8 or ZT4-ZT8). These effects persist in cells pretreated with pertussis toxin (PTX) and, like dopamine, may work to reinforce long-term circadian fluctuations in CNGCs driven by oscillators within the photoreceptors themselves. In contrast, a 15 min exposure to SS28 caused a seemingly paradoxical increase in the sensitivity of CNGCs to cGMP during the early day (ZT4-ZT6), but only in cones maintained in LD. This effect of SS28 desensitizes rapidly, is blocked by pretreatment with PTX, and is selectively mimicked by the cyclohexapeptide agonist MK-678. This transient response also requires activation of phospholipase C and protein kinase C. The transient response to SS28 may play a role in photoreceptor adaptation to rapid changes in ambient illumination. These data also show that photoreceptor responses to at least some peptide neurotransmitters depend on the previous history of light exposure.

Circadian regulation of retinoschisin in the chick retina.

PURPOSE: To investigate the circadian regulation and acute illumination effects on the expression and secretion of retinoschisin from vertebrate retinas. METHODS: Retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12-hour light/12-hour dark (LD) cycles in ovo or in vitro. Quantitative real-time PCR and Western immunoblotting were used to examine the mRNA and protein expressions of retinoschisin at different circadian time points. Pharmacologic treatments in whole retina and dissociated retinal cell cultures were used to investigate the cellular mechanisms underlying the circadian regulation of retinoschisin content and secretion. Different illumination conditions were given to examine changes in retinoschisin content in association with acute light/dark adaptation. RESULTS: The mRNA level, protein expression, and secretion of retinoschisin were under circadian control, all of which were higher at night and lower during the day. The Ras, MAP kinase Erk, CaMKII pathway served as part of the circadian output regulating the rhythmicity of retinoschisin. Blockage of L-type VGCCs dampened the retinoschisin rhythm, but inhibition of L-type VGCCs did not completely abolish the secretion of retinoschisin. The protein expression of retinoschisin also responded to acute illumination changes. CONCLUSIONS: The mRNA and protein expression, as well as retinoschisin secretion, are under circadian control. L-type VGCCs play a role in the circadian regulation of retinoschisin, but the molecular mechanism underlying retinoschisin secretion does not depend on L-type VGCCs. Protein expression of retinoschisin in response to acute illumination changes depends on previous light exposure experience.

Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors.

Energy expenditure and metabolism in the vertebrate retina are under circadian control, as we previously reported that the overall retinal ATP content and various signaling molecules related to metabolism display daily or circadian rhythms. Changes in the fission and fusion process of mitochondria, the major organelles producing ATP, in retinal photoreceptors are largely dependent on light exposure, but whether mitochondrial dynamics in photoreceptors and retinal neurons are under circadian control is not clear. Herein, we investigated the possible roles of circadian oscillators in regulating mitochondrial dynamics, mitophagy, and redox states in the chicken retina and mammalian photoreceptors. After entrainment to 12:12-h light-dark (LD) cycles for several days followed by free-running in constant darkness (DD), chicken embryonic retinas and cone-derived 661W cells were collected in either LD or DD at 6 different zeitgeber time (ZT) or circadian time (CT) points. The protein expression of mitochondrial dynamin-related protein 1 (DRP1), mitofusin 2 (MFN2), and PTEN-induced putative kinase 1 (PINK1) displayed daily rhythms, but only DRP1 was under circadian control in the chicken retinas and cultured 661W cells. In addition, cultured chicken retinal cells responded to acute oxidative stress differently from 661W cells. Using pMitoTimer as a mitochondrial redox indicator, we found that the mitochondrial redox states were more affected by light exposure than regulated by circadian oscillators. Thus, this study demonstrates that the influence of cyclic lights might outweigh the circadian regulation of complex mitochondrial dynamics in light-sensing retinal cells.