Use of SRPK1 inhibitors for the treatment of Diabetic Retinopathy

Microvascular damage results from ischaemia-driven production of pro- angiogenic vascular endothelial growth factor (VEGF). Proximally spliced VEGF is upregulated in the ischemic diabetic retina and has been implicated as the principal driver of the pathological growth and leakage of blood vessels during diabetic retinopathy (DR). Serine-Arginine Rich protein kinase-1 (SRPK1) regulates splicing of VEGF, and inhibition of this kinase with small molecular weight inhibitors has been shown to inhibit choroidal neovascularization (CNV) in mice by decreasing pro-angiogenic and increasing anti-angiogenic VEGF isoforms. These isoforms have previously been described to inhibit increased vascular permeability with protective effects against DR-induced pathology. SRPK1 inhibitors such as SPHINX31 may therefore switch splicing in DR and prevent increased vascular permeability. Retinal pigment epithelial cells were exposed to hyperglycaemia (HG) and hypoxia (Hx) and treated with SPHINX31. SRSF1 localisation in the nuclear speckles, SRPK1 activity and monolayer permeability were assessed by immunofluorescence and Electrical Cell Impedance Sensing. In a rodent model of type 1 diabetes fluorescein fundus angiography (FFA) and optical coherence tomography (OCT) was performed weekly from day 0 to 28. Animals received twice daily topical eye drops with eye formulation control buffer or SPHINX31. On day 1 animals received a single dose of streptozotocin to induce type I diabetes. FFA was quantified using ImageJ; the intensity of sodium fluorescein in the retinal interstitial area and the retinal vessels were measured and the permeability assessed from this relationship. An FFA and OCT time course was used to determine an estimate of permeability and retinal thickness. Retina petals were stained with IB4 and for junctional proteins to deduce vascular density. HG and Hx induced a significant increase (p<0.05) in SRSF1 nuclear localisation, which was blocked by SPHINX31. HG induced a release of SRSF1 from the nuclear speckles (p=0.002). Inhibition of SRPK1 decreased RPE monolayer permeability (p<0.05). The increase in retinal permeability on days 14-28 seen in the diabetic eye formulation only control cohort (n=8) was stabilised following topical treatment of diabetic animals with SPHINX31 (n=9) for 28 days (p<0.0001). Mean retinal thickness increased in diabetes (p<0.05) and this increase appeared to be blocked following SPHINX31 treatment. SPHINX31 protected the retinal barrier from hyperglycaemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. SPHINX31 may therefore be a potential topical therapeutic for DR

Use of SRPK1 inhibitors for the treatment of Diabetic Retinopathy

Microvascular damage results from ischaemia-driven production of pro- angiogenic vascular endothelial growth factor (VEGF). Proximally spliced VEGF is upregulated in the ischemic diabetic retina and has been implicated as the principal driver of the pathological growth and leakage of blood vessels during diabetic retinopathy (DR). Serine-Arginine Rich protein kinase-1 (SRPK1) regulates splicing of VEGF, and inhibition of this kinase with small molecular weight inhibitors has been shown to inhibit choroidal neovascularization (CNV) in mice by decreasing pro-angiogenic and increasing anti-angiogenic VEGF isoforms. These isoforms have previously been described to inhibit increased vascular permeability with protective effects against DR-induced pathology. SRPK1 inhibitors such as SPHINX31 may therefore switch splicing in DR and prevent increased vascular permeability. Retinal pigment epithelial cells were exposed to hyperglycaemia (HG) and hypoxia (Hx) and treated with SPHINX31. SRSF1 localisation in the nuclear speckles, SRPK1 activity and monolayer permeability were assessed by immunofluorescence and Electrical Cell Impedance Sensing. In a rodent model of type 1 diabetes fluorescein fundus angiography (FFA) and optical coherence tomography (OCT) was performed weekly from day 0 to 28. Animals received twice daily topical eye drops with eye formulation control buffer or SPHINX31. On day 1 animals received a single dose of streptozotocin to induce type I diabetes. FFA was quantified using ImageJ; the intensity of sodium fluorescein in the retinal interstitial area and the retinal vessels were measured and the permeability assessed from this relationship. An FFA and OCT time course was used to determine an estimate of permeability and retinal thickness. Retina petals were stained with IB4 and for junctional proteins to deduce vascular density. HG and Hx induced a significant increase (p<0.05) in SRSF1 nuclear localisation, which was blocked by SPHINX31. HG induced a release of SRSF1 from the nuclear speckles (p=0.002). Inhibition of SRPK1 decreased RPE monolayer permeability (p<0.05). The increase in retinal permeability on days 14-28 seen in the diabetic eye formulation only control cohort (n=8) was stabilised following topical treatment of diabetic animals with SPHINX31 (n=9) for 28 days (p<0.0001). Mean retinal thickness increased in diabetes (p<0.05) and this increase appeared to be blocked following SPHINX31 treatment. SPHINX31 protected the retinal barrier from hyperglycaemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. SPHINX31 may therefore be a potential topical therapeutic for DR

Activation of Notch signalling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway

© 2019 the American Physiological Society. The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A

Non‐invasive measurement of retinal permeability in a diabetic rat model

Objective: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high throughput and cost effective alternative technique that minimizes animal usage. Methods: Sodium fluorescein fundus angiography was performed in non- and diabetic Brown Norway rats on days 0, 7, 14, 21 and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post study eyes were fixed, dissected and stained (isolectin B4) to measure required parameters for permeability quantification including: Total vessel length per retinal volume, radius and thickness. Results: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from day 14 to 28 (p [less than] 0.01, p [less than] 0.001, p [less than] 0.0001). Conclusions: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive and cost-effective alternative to the Evans blue assay

Genetic Deletion of the <i>LINC00520</i> Homolog in Mouse Aggravates Angiotensin II-Induced Hypertension

(1) Background: Hypertension is a complex, multifactorial disease that is caused by genetic and environmental factors. Apart from genetic predisposition, the mechanisms involved in this disease have yet to be fully understood. We previously reported that LEENE (lncRNA enhancing endothelial nitric oxide expression, transcribed from LINC00520 in the human genome) regulates endothelial cell (EC) function by promoting the expression of endothelial nitric oxide synthase (eNOS) and vascular growth factor receptor 2 (VEGFR2). Mice with genetic deletion of the LEENE/LINC00520 homologous region exhibited impaired angiogenesis and tissue regeneration in a diabetic hindlimb ischemia model. However, the role of LEENE in blood pressure regulation is unknown. (2) Methods: We subjected mice with genetic ablation of leene and wild-type littermates to Angiotensin II (AngII) and monitored their blood pressure and examined their hearts and kidneys. We used RNA-sequencing to identify potential leene-regulated molecular pathways in ECs that contributed to the observed phenotype. We further performed in vitro experiments with murine and human ECs and ex vivo experiments with murine aortic rings to validate the select mechanism. (3) Results: We identified an exacerbated hypertensive phenotype of leene-KO mice in the AngII model, evidenced by higher systolic and diastolic blood pressure. At the organ level, we observed aggravated hypertrophy and fibrosis in the heart and kidney. Moreover, the overexpression of human LEENE RNA, in part, restored the signaling pathways impaired by leene deletion in murine ECs. Additionally, Axitinib, a tyrosine kinase inhibitor that selectively inhibits VEGFR suppresses LEENE in human ECs. (4) Conclusions: Our study suggests LEENE as a potential regulator in blood pressure control, possibly through its function in ECs

Regulation of nuclear transcription by mitochondrial RNA in endothelial cells

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA–promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial–nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome