Discovery of highly potent and selective KRASG12C degraders by VHL-recruiting PROTACs for the treatment of tumors with KRASG12C-Mutation.

Although several covalent KRASG12C inhibitors have made great progress in the treatment of KRASG12C-mutant cancer, their clinical applications are limited by adaptive resistance, motivating novel therapeutic strategies. Through drug design and structure optimization, a series of highly potent and selective KRASG12C Proteolysis Targeting Chimeras (PROTACs) were developed by incorporating AMG510 and VHL ligand VH032. Among them, degrader YN14 significantly inhibited KRASG12C-dependent cancer cells growth with nanomolar IC50 and DC50 values, and ＞ 95 % maximum degradation (Dmax). Molecular dynamics (MD) simulation showed that YN14 induced a stable KRASG12C: YN14: VHL ternary complex with low binding free energy (ΔG). Notably, YN14 led to tumor regression with tumor growth inhibition (TGI%) rates more than 100 % in the MIA PaCa-2 xenograft model with well-tolerated dose-schedules. We also found that KRASG12C degradation exhibited advantages in overcoming adaptive KRASG12C feedback resistance over KRASG12C inhibition. Furthermore, combination of RTKs, SHP2, or CDK9 inhibitors with YN14 exhibited synergetic efficacy in KRASG12C-mutant cancer cells. Overall, these results demonstrated that YN14 holds exciting prospects for the treatment of tumors with KRASG12C-mutation and boosted efficacy could be achieved for greater clinical applications via drug combination.

Application of Dynamic Enhanced Magnetic Resonance Imaging Texture Analysis Combined with ADCs in Predicting Pelvic Lymph Metastasis of Prostate Cancer.

OBJECTIVE: The application value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) texture analysis combined with apparent diffusion coefficient (ADC) in predicting pelvic lymph node metastasis of prostate cancer was explored. METHODS: The clinical and imaging data of 151 patients with prostate cancer admitted to The Affiliated Tumor Hospital of Guizhou Medical University from November 2019 to November 2021 were retrospectively analysed. According to the final pathological diagnosis results, they were divided into two groups: Metastasis group (n = 63, pelvic lymph node metastasis) and non-metastasis group (n = 88, no pelvic lymph node metastasis). The DCE-MRI texture parameters and ADCs of the two groups were compared using Omni-Kinetics software and MADC software packages. The receiver operating characteristic (ROC) curve was used in evaluating the predictive value of each method and their combination, and Spearman rank correlation analysis was used in evaluating their correlation. RESULTS: The volume transfer (Ktrans) and interstitium-to-plasmarate rate constant (Kep) in the metastatic group were significantly higher than those in the non-metastatic group (p < 0.001). However, no significant difference in extravascular extracellular space volume fraction (Ve) was found between the groups (p > 0.05). The ADC of the metastatic group was lower (p < 0.001). The Ktrans and Kep values were positively correlated with pelvic lymph node metastasis of prostate cancer (r = 0.580, 0.684; p < 0.001), and the ADC was negatively correlated with pelvic lymph node metastasis of prostate cancer (r = -0.478; p < 0.001). The ROC curve showed that the area under the curve (AUC) of DCE-MRI texture analysis parameters Ktrans and Kep combined with ADC was large, and the prediction efficiency increased. The AUC, sensitivity and specificity were 0.974, 95.20% and 93.20% (p < 0.001), respectively. CONCLUSIONS: DCE-MRI texture analysis combined with ADC value can accurately predict pelvic lymphatic metastasis of prostate cancer, which is helpful for the selection and formulation of clinical treatment plans and has certain guiding value for the implementation of pelvic lymph node clearing in patients.

Application of Dynamic Enhanced Magnetic Resonance Imaging Texture Analysis Combined with ADCs in Predicting Pelvic Lymph Metastasis of Prostate Cancer

Objective: The application value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) texture analysis combined with apparent diffusion coefficient (ADC) in predicting pelvic lymph node metastasis of prostate cancer was explored. Methods: The clinical and imaging data of 151 patients with prostate cancer admitted to The Affiliated Tumor Hospital of Guizhou Medical University from November 2019 to November 2021 were retrospectively analysed. According to the final pathological diagnosis results, they were divided into two groups: Metastasis group (n = 63, pelvic lymph node metastasis) and non-metastasis group (n = 88, no pelvic lymph node metastasis). The DCE-MRI texture parameters and ADCs of the two groups were compared using Omni-Kinetics software and MADC software packages. The receiver operating characteristic (ROC) curve was used in evaluating the predictive value of each method and their combination, and Spearman rank correlation analysis was used in evaluating their correlation. Results: The volume transfer (Ktrans) and interstitium-to-plasmarate rate constant (Kep) in the metastatic group were significantly higher than those in the non-metastatic group (p < 0.001). However, no significant difference in extravascular extracellular space volume fraction (Ve) was found between the groups (p > 0.05). The ADC of the metastatic group was lower (p < 0.001). The Ktrans and Kep values were positively correlated with pelvic lymph node metastasis of prostate cancer (r = 0.580, 0.684; p < 0.001), and the ADC was negatively correlated with pelvic lymph node metastasis of prostate cancer (r = −0.478; p < 0.001) (AU)

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations.

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.

Study on microstructure and extinction characteristics of particulate matter in diesel engine fueled with different biodiesels.

Biodiesel combustion particulate matter (PM) is different from diesel combustion PM in terms of microscopic morphology, which directly affects the optical properties of PM. To investigate the effect of the microstructure of biodiesel PM on the extinction characteristics, an experiment was performed on a high-pressure common rail diesel engine to collect PM from three kinds of biodiesel (the main raw materials were soybean oil methyl eater (SME), palm oil methyl eater (PME), and waste cooking oil methyl eater (WME), respectively). The particle size distribution, micro morphology, and extinction characteristics of biodiesel PM were analyzed. Results show that combustion biodiesel reduces PM emissions by up to 84.2%. Compared to PM from diesel, biodiesel PM has a smaller particle size and a higher aggregation degree, which results in weaker light absorption capacity. With the iodine number of biodiesel decreasing, the number concentration of biodiesel PM decreases and the fractal dimension increases, which leads to producing a more complex agglomerate and a consequent reduction in extinction coefficient. The average particle sizes of PM from SME, PME, and WME are 5.1%, 6.7%, and 13.9% lower than that of diesel PM. Compared with diesel combustion PM, the peak absorption coefficients of SME, WME, and PME combustion PM decrease by 8.4%, 11.4%, and 13.3%, respectively. The extinction properties of particles decrease with increasing fractal dimension within the wavelength range of visible light.

Discovery of highly potent and selective CRBN-recruiting EGFRL858R/T790M degraders in vivo.

Currently, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are widely used in the treatment of non-small cell lung cancer (NSCLC). However, the inevitable drug resistance and side effects are the current main obstacle, which motivating novel therapies. Proteolysis targeting chimera (PROTAC), a lately-developed technology to target proteins for degradation, has been utilized for drug development. Therefore, we designed, synthesized and evaluated a series of CRBN-recruiting EGFR degraders. Among them, 13a and 13b significantly inhibited NCI-H1975 cells proliferation with IC50 values of 58.08 nM and 46.82 nM, respectively, whereas exhibited more than 100 µM against A549 or H1299 cells, whose selectivity was more than 1700-fold. 13a and 13b potently induced the EGFRL858R/T790M degradation by ubiquitin proteasome system in a time- and dose-dependent manner but not that of EGFRWT, and the DC50 values of 13b was 13.2 nM, which was the most potent compound in current known CRBN-recruiting EGFRL858R/T790M degraders. 13a and 13b dramatically induced cell apoptosis, cell cycle arrest and inhibited downstream signaling pathways. Furthermore, 13a and 13b effectively and selectively inhibited NCI-H1975 xenograft tumor growth with good pharmacokinetics (PK) properties in vivo. These findings demonstrate that 13a and 13b could serve as candidates for developing the drug for treating NSCLC.

Intraocular lens power calculation after two different successive corneal refractive surgeries.

Purpose: To report two challenging intraocular lens power calculation cases with patients each underwent different successive corneal refractive surgeries, respectively. Observations: Biometry data, including the Back to Front corneal radii ratio (B/F ratio), were collected by Lenstar, IOL Master, and Pentacam AXL for Case 1 (received radial keratotomy (RK) and photorefractive keratectomy (PRK)) and Case 2 (received RK and laser-assisted in situ keratomileusis (LASIK)). The IOL power calculation was determined by several methods, including Shammas, Haigis-L, and Barrett True-K, which are available in the American Society of Cataract and Refractive Surgery online calculator and Pentacam AXL. The Barrett True-K (no history, post-RK) was more accurate in Case 1 (increased B/F ratio), whereas the Shammas, Haigis-L, and Barrett True-K (no history, post-LASIK/PRK) were more accurate in Case 2 (decreased B/F ratio). Conclusion and importance: The B/F ratio may be a factor to be considered when selecting the IOL power calculation formula for patients who undergo two different corneal refractive surgeries. The further study focusing on this issue should be performed to clarify the results in the future.

High-Fat Diet-Induced Functional and Pathologic Changes in Lacrimal Gland.

The lacrimal gland is critical for maintaining the homeostasis of the ocular surface microenvironment through secreting aqueous tears in mammals. Many systemic diseases such as Sjögren syndrome, rheumatoid arthritis, and diabetes can alter the lacrimal gland function, eventually resulting in aqueous tear-deficient dry eye. Here, a high-fat diet (HFD) experimental mouse model was used to clarify how hyperlipidemia affects lacrimal gland function. Aqueous tear secretion fell about 50% after 1 month on a HFD. Lipid droplets accumulated in the matrix and acinar cells of the lacrimal gland after this period, along with changes in the lipid metabolism, changes in gene expression levels, and disruption of fatty acid oxidative activity. Immune cell infiltration and rises in the gene expression levels of the inflammation-related cytokines Il1ß, Tnfα, Tsg6, Il10, Mmp2, and Mmp9 were found. HFD also induced mitochondrial hypermegasoma, increased apoptosis, and decreased lacrimal gland acinar cell proliferation. Replacement of the HFD with the standard diet partially reversed pathologic changes in the lacrimal gland. Similarly, supplementing the HFD with fenofibrate also partially reversed the inhibited tear secretion and reduced lipid accumulation, inflammation, and oxidative stress levels. The authors conclude that a HFD induces pathophysiological changes and functional decompensation of the lacrimal gland. Therefore, ingestion of a HFD may be a causative factor of dry eye disease.

A Novel Tissue-Engineered Corneal Stromal Equivalent Based on Amniotic Membrane and Keratocytes.

Purpose: To investigate a novel strategy in constructing tissue-engineered corneal stromal equivalent based on amniotic membrane and keratocytes. Methods: The ultrathin amniotic membrane (UAM) was laminated, with corneal stromal cells (CSCs) distributed between the space of the layered UAMs. Calcein AM staining was used to evaluate cellular viability, morphology, and arrangement. Immunostaining, qRT-PCR, and Western blot were performed to detect gene and protein expression in keratocytes. Optical coherence tomography visualized the cross sections and thickness of the UAM construction. The microstructure of the CSC-secreted extracellular matrix (ECM) was investigated by scanning electron microscopy and transmission electron microscopy (TEM). To evaluate the feasibility of the multilayer UAM-CSC lamination for surgery, the corneal substitute was used to perform lamellar keratoplasty. Slit lamp microscopy and corneal fluorescein staining were performed in postsurgery observation. Results: The CSCs maintained their keratocyte phenotype and secreted well-organized ECM on the aligned UAM surface. The four-layer UAM-CSC lamination attained half thickness of the human cornea (250 ± 18 µm) after 8 weeks' culture, which also showed promising optimal transparency. In TEM images, the CSC-generated ECM displayed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea microstructure. Furthermore, the stromal equivalent was successfully preformed in lamellar keratoplasty. Four weeks post surgery, the substitute was well integrated into the recipient cornea and completely epithelialized without myofibroblast differentiation. Conclusions: Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM, which was capable of promoting the development of corneal stroma-like tissues in vitro, establishing a new avenue for basic research and therapeutic potential.

Sleep deprivation disrupts the lacrimal system and induces dry eye disease.

Sleep deficiency is a common public health problem associated with many diseases, such as obesity and cardiovascular disease. In this study, we established a sleep deprivation (SD) mouse model using a 'stick over water' method and observed the effect of sleep deficiency on ocular surface health. We found that SD decreased aqueous tear secretion; increased corneal epithelial cell defects, corneal sensitivity, and apoptosis; and induced squamous metaplasia of the corneal epithelium. These pathological changes mimic the typical features of dry eye. However, there was no obvious corneal inflammation and conjunctival goblet cell change after SD for 10 days. Meanwhile, lacrimal gland hypertrophy along with abnormal lipid metabolites, secretory proteins and free amino-acid profiles became apparent as the SD duration increased. Furthermore, the ocular surface changes induced by SD for 10 days were largely reversed after 14 days of rest. We conclude that SD compromises lacrimal system function and induces dry eye. These findings will benefit the clinical diagnosis and treatment of sleep-disorder-related ocular surface diseases.

Public data mining plus domestic experimental study defined involvement of the old-yet-uncharacterized gene matrix-remodeling associated 7 (MXRA7) in physiopathology of the eye.

Matrix-remodeling associated 7 (MXRA7) gene was first reported in 2002 and named so for its co-expression with several genes known to relate with matrix-remodeling. However, not any studies had been intentionally performed to characterize this gene. We started defining the functions of MXRA7 by integrating bioinformatics analysis and experimental study. Data mining of MXRA7 expression in BioGPS, Gene Expression Omnibus and EurExpress platforms highlighted high level expression of Mxra7 in murine ocular tissues. Real-time PCR was employed to measure Mxra7 mRNA in tissues of adult C57BL/6 mice and demonstrated that Mxra7 was preferentially expressed at higher level in retina, corneas and lens than in other tissues. Then the inflammatory corneal neovascularization (CorNV) model and fungal corneal infections were induced in Balb/c mice, and mRNA levels of Mxra7 as well as several matrix-remodeling related genes (Mmp3, Mmp13, Ecm1, Timp1) were monitored with RT-PCR. The results demonstrated a time-dependent Mxra7 under-expression pattern (U-shape curve along timeline), while all other matrix-remodeling related genes manifested an opposite changes pattern (dome-shape curve). When limited data from BioGPS concerning human MXRA7 gene expression in human tissues were looked at, it was found that ocular tissue was also the one expressing highest level of MXRA7. To conclude, integrative assay of MXRA7 gene expression in public databank as well as domestic animal models revealed a selective high expression MXRA7 in murine and human ocular tissues, and its change patterns in two corneal disease models implied that MXRA7 might play a role in pathological processes or diseases involving injury, neovascularization and would healing.

Descemet's Membrane Supports Corneal Endothelial Cell Regeneration in Rabbits.

Descemet's membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet's membrane. Descemet's membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.

Dry Eye Management: Targeting the Ocular Surface Microenvironment.

Dry eye can damage the ocular surface and result in mild corneal epithelial defect to blinding corneal pannus formation and squamous metaplasia. Significant progress in the treatment of dry eye has been made in the last two decades; progressing from lubricating and hydrating the ocular surface with artificial tear to stimulating tear secretion; anti-inflammation and immune regulation. With the increase in knowledge regarding the pathophysiology of dry eye, we propose in this review the concept of ocular surface microenvironment. Various components of the microenvironment contribute to the homeostasis of ocular surface. Compromise in one or more components can result in homeostasis disruption of ocular surface leading to dry eye disease. Complete evaluation of the microenvironment component changes in dry eye patients will not only lead to appropriate diagnosis, but also guide in timely and effective clinical management. Successful treatment of dry eye should be aimed to restore the homeostasis of the ocular surface microenvironment.

ROS-mediated Different Homeostasis of Murine Corneal Epithelial Progenitor Cell Line under Oxidative Stress.

The role of ROS in stem cell biology has not been fully illustrated and understood. Here we compared the different responses and investigated the mechanism underlying oxidative stress induced by hydrogen peroxide (H2O2) between murine corneal epithelial progenitor cell line (TKE2) and mature murine corneal epithelial cells (MCE). TKE2 showed a different homeostasis and strong resistance to H2O2. TKE2 reduced the production of ROS, inhibited ROS generation enzyme NADPH oxidase 4 (NOX4), and increased dual specificity phosphatase 6 (DUSP6). Furthermore, TKE2 activated nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway, regulated miR-125B1 and miR-29B1, and elevated levels of antioxidants glutathione S-transferase P (GSTP) and superoxide dismutases (SOD). The association with ROS of the cells was also verified by RNA interference approach and pharmacological antagonization. In addition, TKE2 enhanced the autophagy after exposure to H2O2. The novel evidence suggests that TKE2 cells have different homeostasis and strong antioxidant properties against oxidative stress via the regulation of ROS formation and pathway.

An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering.

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 µm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Serum amyloid A and pairing formyl peptide receptor 2 are expressed in corneas and involved in inflammation-mediated neovascularization.

AIM: To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice. METHODS: Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting. RESULTS: Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV. CONCLUSION: SAA-FPR2 pathway composing genes were expressed in normal murine corneas and, upon inflammatory stimuli challenge to the corneas, their expressions were up-regulated, suggesting their roles in pathogenesis of CorNV. The potential usefulness of SAA-FPR2 targets in future management of CorNV-related diseases deserves investigation.

IL-17 plays a central role in initiating experimental Candida albicans infection in mouse corneas.

The pathogenesis of fungal infection in the cornea remains largely unclear. To understand how the immune system influences the progression of fungal infection in corneas, we inoculated immunocompetent BALB/c mice, neutrophil- or CD4⁺ T-cell-depleted BALB/c mice, and nude mice with Candida albicans. We found that only immunocompetent BALB/c mice developed typical Candida keratitis (CaK), while the other mouse strains lacked obvious clinical manifestations. Furthermore, CaK development was blocked in immunocompetent mice treated with anti-IL-17A or anti-IL-23p19 to neutralize IL-17 activity. However, no significant effects were observed when Treg cells, γδ T cells, or IFN-γ were immunodepleted. Upon infection, the corneas of BALB/c mice were infiltrated with IL-17-producing leukocytes, including neutrophils and, to a lesser degree, CD4⁺ T cells. In contrast, leukocyte recruitment to corneas was significantly diminished in nude mice. Indeed, nude mice produced much less chemokines (e.g. CXCL1, CXCL2, CXCL10, CXCL12, CCL2, and IL-6) in response to inoculation. Remarkably, addition of CXCL2 during inoculation restored CaK induction in nude mice. In contrast to its therapeutic effect on CaK, neutralization of IL-17 exacerbated Candida-induced dermatitis in skin. We conclude that IL-17, mainly produced by neutrophils and CD4⁺ T cells in the corneas, is essential in the pathogenesis of CaK.

Profiling of genes associated with the murine model of oxygen-induced retinopathy.

PURPOSE: To compare the clinical features and gene expression patterns of the physiologic development of retinal vessels and oxygen-induced retinopathy (OIR) in a mouse model, with the aim of identifying differential regulators of physiologic and pathological angiogenesis in the retina. METHODS: C57BL/6J mice were used. Seven-day-old pups were subjected to OIR induction following the standard protocols of entering a hyperoxic chamber on day 7 (P7) and returning to a normoxic condition (relative hypoxia) on day 12 (P12). The retinal vasculatures in the OIR model 24 h (P8-O) or 5 days (P12-O) after switching to the hyperoxic environment and 24 h (P13-O) after returning to normoxic conditions were evaluated with retinal flat mounts and compared with those of age-matched controls (i.e., P8-N, P12-N, P13-N). Gene expression profiling was performed using Phalanx Mouse Whole Genome OneArray microarrays. Normal 9-day-old mice were considered representative of physiologic angiogenesis and compared with 30-day-old mice. A bioinformatics analysis was performed on differentially expressed genes using various comparisons, and real-time reverse-transcription PCR was used to confirm the changes in the genes of interest. RESULTS: The sequential orders and patterns of vasculature development in normal mice and the OIR models were significantly different. In brief, in the early days (P1 to P7) for normal mice, retinal vessels grew from the optic disc into the non-vascularized retina in a radial fashion. In the hyperoxic stage of the OIR model, the main central retina became devoid of a vascular network, and when the mice returned to the normoxic room, the vessels grew from peripheral perfused areas toward the center of the retina, but the development of intermediate and deep layers of vasculature was significantly delayed. Gene profiling at three critical time points (P8, P12, and P13) showed that 162 probes were upregulated to ≥1.5-fold or downregulated to ≤0.67-fold at one or more time points in the OIR model compared to the controls. In the 45 upregulated genes for the P8-O/P8-N group, enriched genes were mainly related to cytoskeleton formation, whereas the 62 upregulated genes for P13-O/P13-N participated in various pathological processes. In the physiologic conditions on P9, however, 135 genes were upregulated compared with P30; the gap junction and Fc gamma R-mediated phagocytosis were the two main enriched pathways for these genes. Fifty-three probes, including vascular endothelium growth factor A, annexin A2, and endothelin 2, changed at P13-O but not at P9-N, and these changed genes might reflect the modulation of pathological neovascularization. CONCLUSIONS: Angiogenesis in physiologic and pathological conditions is characterized by the differential presentation of vasculature and gene expression patterns. Investigation of those genes unique to the OIR model may help develop new strategies and therapies for intervening in retinal neovascularization.

Low concentration of S100A8/9 promotes angiogenesis-related activity of vascular endothelial cells: bridges among inflammation, angiogenesis, and tumorigenesis?

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 µg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 µg/mL S100A8 revealed that ribosome pathway, pathogenic Escherichia coli infection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

αA-crystallin in the pathogenesis and intervention of experimental murine corneal neovascularization.

This study was to determine the potential roles of lens crystallins in the pathogenesis of corneal neovascularization (CorNV) and implications in therapy of CorNV-related diseases. Suture- or chemical burn-induced CorNV in different strains of mice were used. Changes of gene expression patterns were analyzed by microarray, and the results of interesting genes were confirmed by real-time quantitative PCR and Western blot. Mice deficient in αA-crystallin gene were used to evaluate the role of αA-crystallin in the development of CorNV. In some animals, exogenous αA-crystallin proteins were injected around time of CorNV induction. CorNV was assessed by slit-lamp, flat-mounts and histology. In BALB/C mice, the expression of α-, ß-, and γ-crystallins were up-regulated at day 5 and returned to baseline level at day 10 of suture-induced CorNV, but remained up-regulated from day 6 to day 14 of chemical burn-induced CorNV. In chemical burn-induced CorNV in C57BL/6J mice, however, they were down-regulated at day 6. Corneal crystallins were down-regulated in both CorNV models at all time points in both BALB/c and C57BL/6J mice. Comparison of CorNV development in αA-crystallin-deficient mice and that in wild-type mice revealed no significant difference. Subconjunctival injection of αA-crystallin significantly attenuated suture-induced CorNV, and the inhibitory activity might be implemented by the increased expression of soluble VEGFR-1. In conclusion, the expression patterns of lens crystallins were time- and strain-dependent but different from that of corneal crystallins in mouse CorNV models. Exogenous αA-crystallin protein attenuated CorNV, potentially by increasing the expression of soluble VEGFR-1.