ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects.

Transforming growth factor ß (TGFß) is present in blood of patients who do not respond to anti-programmed cell death (ligand) 1 [PD-(L)1] treatment, and through synergy with vascular endothelial growth factor (VEGF), it helps to create an environment that promotes tumor immune evasion and immune tolerance. Therefore, simultaneous inhibition of TGFß/VEGF is more effective than targeting TGFß alone. In this study, the dual inhibitory mechanism of TU2218 was identified through in vitro analysis mimicking the tumor microenvironment, and its antitumor effects were analyzed using mouse syngeneic tumor models. TU2218 directly restored the activity of damaged cytotoxic T lymphocytes (CTLs) and natural killer cells inhibited by TGFß and suppressed the activity and viability of regulatory T cells. The inactivation of endothelial cells induced by VEGF stimulation was completely ameliorated by TU2218, an effect not observed with vactosertib, which inhibits only TGFß signaling. The combination of TU2218 and anti-PD1 therapy had a significantly greater antitumor effect than either drug alone in the poorly immunogenic B16F10 syngeneic tumor model. The mechanism of tumor reduction was confirmed by flow cytometry, which showed upregulated VCAM-1 expression in vascular cells and increased influx of CD8 + CTLs into the tumor. As another strategy, combination of anti-CTLA4 therapy and TU2218 resulted in high complete regression (CR) rates in CT26 and WEHI-164 tumor models. In particular, immunological memory generated by the combination of anti-CTLA4 and TU2218 in the CT26 model prevented the development of tumors after additional tumor cell transplantation, suggesting that the TU2218-based combination has therapeutic potential in immunotherapy.

Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.

Anti-proliferation evaluation of new derivatives of indole-6-carboxylate ester as receptor tyrosine kinase inhibitors.

Aim: The main goal was to create two new groups of indole derivatives, hydrazine-1-carbothioamide (4a and 4b) and oxadiazole (5, and 6a-e) that target EGFR (4a, 4b, 5) or VEGFR-2 (6a-e). Materials & methods: The new derivatives were characterized using various spectroscopic techniques. Docking studies were used to investigate the binding patterns to EGFR/VEGFR-2, and the anti-proliferative properties were tested in vitro. Results: Compounds 4a (targeting EGFR) and 6c (targeting VEGFR-2) were the most effective cytotoxic agents, arresting cancer cells in the G2/M phase and inducing the extrinsic apoptosis pathway. Conclusion: The results of this study show that compounds 4a and 6c are promising cytotoxic compounds that inhibit the tyrosine kinase activity of EGFR and VEGFR-2, respectively.

[Box: see text].

Immunohistochemistry Screening of Different Tyrosine Kinase Receptors in Canine Solid Tumors-Part I: Proposal of a Receptor Panel to Predict Therapies.

The use of tyrosine kinase inhibitors (TKI) has been growing in veterinary oncology and in the past few years several TKI have been tested in dogs. However, different from human medicine, we lack strategies to select patients to be treated with each TKI. Therefore, this study aimed to screen different tumor subtypes regarding TKI target immunoexpression as a predictor strategy to personalize the canine cancer treatment. It included 18 prostatic carcinomas, 36 soft tissue sarcomas, 20 mammary gland tumors, 6 urothelial bladder carcinomas, and 7 tumors from the endocrine system. A total of 87 patients with paraffin blocks were used to perform immunohistochemistry (IHC) of human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptors 1 (EGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet derived growth factor receptor beta (PDGFR-ß), c-KIT, and extracellular signal-regulated kinase 1/2 (ERK1/ERK2). The immunohistochemical screening revealed a heterogeneous protein expression among histological types with mesenchymal tumors showing the lowest expression level and carcinomas the highest expression. We have demonstrated by IHC screening that HER2, EGFR1, VEGFR-2, PDGFR-ß and ERK1/ERK2 are commonly overexpressed in dogs with different carcinomas, and KIT expression is considered relatively low in the analyzed samples.

Lack of VEGFA/KDR Signaling in Conventional Renal Cell Carcinoma Explains the Low Efficacy of Target Therapy and Frequent Adverse Events.

It is acknowledged that conventional renal cell carcinoma (cRCC), which makes up 85% of renal malignancies, is a highly vascular tumor. Humanized monoclonal antibodies were developed to inhibit tumor neo-angiogenesis, which is driven by VEGFA/KDR signaling. The results largely met our expectations, and in several cases, adverse events occurred. Our study aimed to analyze the expression of VEGFA and its receptor KDR by immunohistochemistry in tissue multi-array containing 811 cRCC and find a correlation between VEGFA/KDR signaling and new vessel formation. None of the 811 cRCC displayed VEGFA-positive immunostaining. However, each glomerulus in normal kidney showed VEGFA-positive endothelial cells. KDR expression in endothelial meshwork was found in only 9% of cRCC, whereas 2% of the cRCC displayed positive KDR reaction in the cytoplasm of tumor cells. Our results disclose the involvement of VEGFA/KDR signaling in the neo-vascularization of cRCC and explain the frequent resistance to drugs targeting the VEGFA/KDR signaling and the high frequency of adverse events.

Antitumor activity of anlotinib in malignant melanoma: modulation of angiogenesis and vasculogenic mimicry.

Malignant melanoma presents a formidable challenge due to its aggressive metastatic behavior and limited response to current treatments. To address this, our study delves into the impact of anlotinib on angiogenesis and vasculogenic mimicry using malignant melanoma cells and human umbilical vein endothelial cells. Evaluating tubular structure formation, cell proliferation, migration, invasion, and key signaling molecules in angiogenesis, we demonstrated that anlotinib exerts a dose-dependent inhibition on tubular structures and effectively suppresses cell growth and invasion in both cell types. Furthermore, in a mouse xenograft model, anlotinib treatment resulted in reduced tumor growth and vascular density. Notably, the downregulation of VEGFR-2, FGFR-1, PDGFR-ß, and PI3K underscored the multitargeted antitumor activity of anlotinib. Our findings emphasize the therapeutic potential of anlotinib in targeting angiogenesis and vasculogenic mimicry, contributing to the development of novel strategies for combating malignant melanoma.

Targeted Delivery of Diphtheria Toxin into VEGFR1/VEGFR2 Overexpressing Cells Induces Anti-angiogenesis Activity.

BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.

Molecular docking aided machine learning for the identification of potential VEGFR inhibitors against renal cell carcinoma.

Renal cell carcinoma is a highly vascular tumor associated with vascular endothelial growth factor (VEGF) expression. The Vascular Endothelial Growth Factor -2 (VEGF-2) and its receptor was identified as a potential anti-cancer target, and it plays a crucial role in physiology as well as pathology. Inhibition of angiogenesis via blocking the signaling pathway is considered an attractive target. In the present study, 150 FDA-approved drugs have been screened using the concept of drug repurposing against VEGFR-2 by employing the molecular docking, molecular dynamics, grouping data with Machine Learning algorithms, and density functional theory (DFT) approaches. The identified compounds such as Pazopanib, Atogepant, Drosperinone, Revefenacin and Zanubrutinib shown the binding energy - 7.0 to - 9.5 kcal/mol against VEGF receptor in the molecular docking studies and have been observed as stable in the molecular dynamic simulations performed for the period of 500 ns. The MM/GBSA analysis shows that the value ranging from - 44.816 to - 82.582 kcal/mol. Harnessing the machine learning approaches revealed that clustering with K = 10 exhibits the relevance through high binding energy and satisfactory logP values, setting them apart from compounds in distinct clusters. Therefore, the identified compounds are found to be potential to inhibit the VEGFR-2 and the present study will be a benchmark to validate the compounds experimentally.

Identification of Benzothiazoles Bearing 1,3,4-Thiadiazole as Antiproliferative Hybrids Targeting VEGFR-2 and BRAF Kinase: Design, Synthesis, BIO Evaluation and In Silico Study.

Cancer remains a leading cause of death worldwide, often resulting from uncontrolled growth in various organs. Protein kinase inhibitors represent an important class of targeted cancer therapies. Recently, the kinases BRAF and VEGFR-2 have shown synergistic effects on tumor progression. Seeking to develop dual BRAF/VEGFR-2 inhibitors, we synthesized 18 amino-benzothiazole derivatives with structural similarities to reported dual inhibitors. Four compounds-4a, 4f, 4l, and 4r-demonstrated remarkable cytotoxicity, with IC50 values ranging from 3.58 to 15.36 µM, against three cancer cell lines. Furthermore, these compounds showed IC50 values of 38.77-66.22 µM in the case of a normal cell line, which was significantly safer than the reference, sorafenib. Subsequent investigation revealed that compound 4f exhibited the capacity to inhibit the BRAF and VEGFR-2 enzymes, with IC50 values similar to sorafenib (0.071 and 0.194 µM, respectively). Moreover, compound 4f caused G2-M- and S-phase cycle arrest. Molecular modeling demonstrated binding patterns compatible with inhibition for both targets, where 4f exerted the critical interactions in the BRAF site and interacted in the VEGFR-2 site in a manner akin to sorafenib, demonstrating affinity similar to dabrafenib.

Mtnr1b deletion disrupts placental angiogenesis through the VEGF signaling pathway leading to fetal growth restriction.

The placenta, as a "transit station" between mother and fetus, has functions delivering nutrients, excreting metabolic wastes and secreting hormones. A healthy placenta is essential for fetal growth and development while the melatonergic system seems to play a critical physiological role in this organ since melatonin, its synthetic enzymes and receptors are present in the placenta. In current study, Mtnr1a and Mtnr1b knockout mice were constructed to explore the potential roles of melatonergic system played on the placental function and intrauterine growth retardation (IUGR). The result showed that Mtnr1a knockout had little effect on placental function while Mtnr1b knockout reduced placental efficiency and increased IUGR. Considering the extremely high incidence of IURG in sows, the pregnant sows were treated with melatonin. This treatment reduced the incidence of IUGR. All the evidence suggests that the intact melatonergic system in placenta is required for its function. Mechanistical studies uncovered that Mtnr1b knockout increased placental oxidative stress and apoptosis but reduced the angiogenesis. The RNA sequencing combined with histochemistry study identified the reduced angiogenesis and placental vascular density in Mtnr1b knockout mice. These alterations were mediated by the disrupted STAT3/VEGFR2/PI3K/AKT pathway, i.e., Mtnr1b knockout reduced the phosphorylation of STAT3 which is the promotor of VEGFR2. The downregulated VEGFR2 and its downstream elements of PI3K and AKT expressions, then, jeopardizes the angiogenesis and placental development.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.

Pimitespib, a Novel Heat Shock Protein 90 Inhibitor, Is Effective in Treating Renal Cell Carcinoma by Anti-angiogenetic Signaling.

BACKGROUND/AIM: Most clear cell renal cell carcinomas (ccRCCs) have a dysfunctional von Hippel-Lindau tumor suppressor protein (VHL). Hypoxia-inducible factors 1 and 2 alpha (HIF1α and HIF2α) accumulate in ccRCC with dysfunctional VHL and up-regulate the vascular endothelial growth factor (VEGF) pathway and tumor angiogenesis. Recently, pimitespib (PIM), a potent ATP-competitive inhibitor of heat shock protein 90 (HSP90), was developed. PIM down-regulates the expression of HIF, a key protein in ccRCC progression, with anti-angiogenic effects. This study aimed to examine the effectiveness of PIM in ccRCC and the underlying mechanisms. MATERIALS AND METHODS: The efficacy and mechanism of PIM against ccRCCs was evaluated using ccRCC cell lines. RESULTS: PIM inhibited the VEGFR pathway by down-regulating VEGFR 2, phosphorylated VEGFR 2, and protein levels in downstream signaling pathways. The growth of ccRCC cell lines was inhibited by PIM. Furthermore, PIM inhibits HIF1α, HIF2α, and VEGF expression, suggesting that PIM may suppress angiogenesis in addition to the VEGFR pathway. CONCLUSION: PIM provides a novel approach for treating ccRCC and holds promise for future clinical strategies. Further in vivo and clinical research is required to elucidate the detailed relationship between the effects of PIM and ccRCC.

MEG3-Mediated Oral Squamous-Cell-Carcinoma-Derived Exosomal miR-421 Activates Angiogenesis by Targeting HS2ST1 in Vascular Endothelial Cells.

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

Variations of VEGFR2 Chemical Space: Stimulator and Inhibitory Peptides.

The kinase pathway plays a crucial role in blood vessel function. Particular attention is paid to VEGFR type 2 angiogenesis and vascular morphogenesis as the tyrosine kinase pathway is preferentially activated. In silico studies were performed on several peptides that affect VEGFR2 in both stimulating and inhibitory ways. This investigation aims to examine the molecular properties of VEGFR2, a molecule primarily involved in the processes of vasculogenesis and angiogenesis. These relationships were defined by the interactions between Vascular Endothelial Growth Factor receptor 2 (VEGFR2) and the structural features of the systems. The chemical space of the inhibitory peptides and stimulators was described using topological and energetic properties. Furthermore, chimeric models of stimulating and inhibitory proteins (for VEGFR2) were computed using the protein system structures. The interaction between the chimeric proteins and VEGFR was computed. The chemical space was further characterized using complex manifolds and high-dimensional data visualization. The results show that a slightly similar chemical area is shared by VEGFR2 and stimulating and inhibitory proteins. On the other hand, the stimulator peptides and the inhibitors have distinct chemical spaces.

A series of quinazolin-4(3H)-one-morpholine hybrids as anti-lung-cancer agents: Synthesis, molecular docking, molecular dynamics, ADME prediction and biological activity studies.

In this study, we synthesized 15 novel quinazoline-morpholinobenzylideneamino hybrid compounds from methyl anthranilate and we assessed their cytotoxicity via in vitro assays against A549 and BEAS-2B cell lines. Molecular docking studies were conducted to evaluate the protein-ligand interactions and inhibition mechanisms on nine different molecular targets, while molecular dynamics (MD) simulations were carried out to assess the stability of the best docked ligand-protein complexes. Additionally, ADME prediction was carried out to determine physicochemical parameters and drug likeness. According to the cytotoxicity assays, compound 1 (IC50 = 2.83 µM) was found to be the most active inhibitor against A549 cells. While the selectivity index (SI) of compound 1 is 29, the SI of the reference drugs paclitaxel and sorafenib, used in this study, are 2.40 and 4.92, respectively. Among the hybrid compounds, 1 has the best docking scores against VEGFR1 (-11.744 kcal/mol), VEGFR2 (-12.407 kcal/mol) and EGFR (-10.359 kcal/mol). During MD simulations, compound 1 consistently exhibited strong hydrogen bond interactions with the active sites of VEGFR1 and 2, and these interactions were maintained for more than 90% of the simulation time. Additionally, the RMSD and RMSF values of the ligand-protein complexes exhibited high stability at their minimum levels around 1-2 Å. In conclusion, these findings suggest that compound 1 may be a potent and selective inhibitor candidate for lung cancer treatment and inhibition of VEGFR2, especially.

Design, synthesis and cytotoxic evaluation of new thieno[2,3-d]pyrimidine analogues as VEGFR-2/AKT dual inhibitors, apoptosis and autophagy inducers.

Novel thieno[2,3-d]pyrimidine analogues were designed, synthesized and evaluated for anti-proliferative activity against HepG-2, PC-3 and MCF-7 cancer cell lines. In addition, WI-38 normal cell line was used to explore the safety of all the tested compounds. Compounds 2 (IC50 = 4.29 µM HePG-2, 10.84 µM MCF-7), 6 (IC50 = 14.86 µM HePG-2, 8.04 µM PC-3 and 12.90 µM MCF-7) and 17 (IC50 = 9.98 µM HePG-2, 33.66 µM PC-3 and 14.62 µM MCF-7) were the most promising candidates on the tested cancer cells with high selective toxicity-sparing normal cells. A further mechanistic evaluation revealed promising kinase inhibitory activity, where compound 2 inhibited VEGFR-2 and AKT at IC50 = 0.161 and 1.06 µM, respectively, Furthermore, derivative 6 inhibited VEGFR-2 and AKT at IC50 = 0.487 and 0.364 µM, respectively, while compound 17 showed IC50 = 0.164 and 0.452 µM, respectively. Moreover, compounds 2, 6 resulted in G1 phase cell cycle arrest while candidate 17 arrest cell cycle at G2/M phase. Similar to the apoptosis results, compound 17 showed the highest autophagic induction among the evaluated derivatives. Finally, docking studies were conducted to assess the binding patterns of these active derivatives. The results showed that the binding patterns inside the active sites of both the VEGFR-2 and AKT-1 (allosteric pocket) crystal structures were identical to the reference ligands.

Identification of an osteopontin-derived peptide that binds neuropilin-1 and activates vascular repair responses and angiogenesis.

The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.

Physicochemical characterization and anti-angiogenesis activity of polysaccharides from Amauroderma rugosum, a medicinal and edible mushroom.

Amauroderma rugosum (AR) is commonly recognized as a medicinal fungus, often used as an alternative to Ganoderma lucidum. There is a scarcity of comprehensive and in-depth research on its bioactive polysaccharides and their associated biological activities. Herein, we isolated the polysaccharide fractions extracted from AR (ARPs) and investigated their primary structure and anti-angiogenic activities, given that various diseases are associated with excessive angiogenesis. Four polysaccharide fractions including ARP-0, ARP-1, ARP-2, and ARP-5 were heteropolysaccharides with different molecular weights, monosaccharide compositions, and micromorphologies, highlighting their varying bioactive profiles. Treatment of human umbilical vein endothelial cells with these polysaccharide fractions showed that only ARP-5 inhibited cell proliferation after vascular endothelial growth factor (VEGF) stimulation. Similarly, ARP-5 inhibited human umbilical vein endothelial cells migration, invasion, and tube formation upon VEGF (50 ng/mL) treatment. Moreover, compared with the insignificant effects of ARP-0, ARP-1, and ARP-2, ARP-5 impeded angiogenesis in zebrafish embryos. Additionally, ARP-5 downregulated the VEGF/VEGFR2 signaling pathway in a dose-dependent manner, suggesting that ARP-5 exerts its anti-angiogenic activities by blocking the VEGF/VEGFR2-mediated angiogenesis signaling pathway. Taken together, the study findings shed light on the primary structure and bioactivity of ARPs.

Characteristics of Retinal Vascular Degeneration and the Expression of Vessel-Related Claudin Proteins in Retinal Degeneration Mouse.

INTRODUCTION: This study aimed to investigate the characteristics of retinal vascular degeneration and the expression of vessel-related claudin (CLD) proteins in retinal degeneration mouse (Pde6ßrd1/rd1 rd1 mouse). METHODS: Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas. RESULTS: Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15. CONCLUSION: Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.

Novel NO-TZDs and trimethoxychalcone-based DHPMs: design, synthesis, and biological evaluation as potential VEGFR-2 inhibitors.

Novel series of nitric oxide-releasing thiazolidine-2,4-diones (NO-TZD-3a-d,5,6) and 3,4,5-trimethoxychalcone-based multifunctional 1,4-dihydropyrimidines (CDHPM-10a-g) have been designed and synthesised as potent broad-spectrum anticancer agents with potential VEGFR-2 inhibition. The designed analogs were evaluated for their anticancer activities towards a full panel of NCI-60 tumour cell lines and CDHPM-10a-g emerged mean %inhibitions ranging from 76.40 to 147.69%. Among them, CDHPM-10e and CDHPM-10f demonstrated the highest MGI% of 147.69 and 140.24%, respectively. Compounds CDHPM-10a,b,d-f showed higher mean %inhibitory activity than the reference drug sorafenib (MGI% = 105.46%). Superiorly, the hybrid CDHPM-10e displayed the highest potencies towards all the herein tested subpanels of nine types of cancer with MGI50 of 1.83 µM. Also, it revealed potent cytostatic single-digit micromolar activity towards the herein examined cancer cell lines. The designed compounds CDHPM-10a-g were exposed as potent non-selective broad-spectrum anticancer agents over all NCI subpanels with an SI range of 0.66-1.97. In addition, the target analog CDHPM-10e revealed potency towards VEGFR-2 kinase comparable to that of sorafenib with a sub-micromolar IC50 value of 0.11 µM. Also, CDHPM-10e could effectively induce Sub-G1-phase arrest and prompt apoptosis via caspase and p53-dependent mechanisms. Furthermore, CDHPM-10e revealed significant anti-metastatic activity as detected by wound healing assay. The modelling study implies that CDHPM-10e overlaid well with sorafenib and formed a strong H-bond in the DFG binding domain. The ADMET studies hinted out that CDHPM-10e met Pfizer's drug-likeness criteria. The presented novel potent anticancer agent merits further devotion as a new lead product in developing more chalcone-based VEGFR-2 inhibitors.