Αvβ3 Expression Research Articles

Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvβ6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvβ6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvβ6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvβ6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvβ6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvβ6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvβ6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvβ6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvβ6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvβ6-positive CCA.

Assessing Doxorubicin-Induced Cardiomyopathy by 99mTc-3PRGD2 Scintigraphy Targeting Integrin αvβ3 in a Rat Model.

The present study evaluated interstitial alterations in doxorubicin-induced cardiomyopathy using a radiolabeled RGD peptide 99mTc-3PRGD2 specific for integrin αvβ3 that targets myofibroblasts.Cardiomyopathy was induced in 20 Sprague-Dawley rats by intraperitoneal doxorubicin injections (2.5 mg/kg/week) for up to six weeks. 99mTc-3PRGD2 scintigraphy was performed in control rats (n = 6) at baseline and three, six, and nine weeks after first doxorubicin administration (n = 6, 6, and 5 for each time point). For another three rats of 6-week modeling, cold c(RGDyK) was co-injected with 99mTc-3PRGD2 to evaluate specific radiotracer binding. Semi-quantitative parameters were acquired to compare radiotracer uptake among all groups. The biodistribution of 99mTc-3PRGD2 was evaluated by a γ-counter after scintigraphy. Haematoxylin and eosin, and Masson's staining were used to evaluate myocardial injury and fibrosis, while western blotting and immunofluorescence co-localization were used to analyze integrin αvβ3 expression in the myocardium.The 99mTc-3PRGD2 half-life in the cardiac region (Heartt 1/2) of the 9-week model and heart radioactivity percentage (%Heart20 min, %Heart40 min and %Heart60 min) of the 6 and 9-week models were significantly increased compared to the control. Heart-to-background ratio (HBR20 min, HBR40 min and HBR60 min) increase began in the third week, continued until the sixth week, and was reversed in the ninth week, which paralleled the changing trend of cardiac integrin αvβ3 expression. The myocardial biodistribution of 99mTc-3PRGD2 was significantly correlated with integrin β3 expression.The 99mTc-3PRGD2 scintigraphy allows for non-invasive visualization of interstitial alterations during doxorubicin-induced cardiomyopathy.

Differential expression of αVβ3 and αVβ6 integrins in prostate cancer progression.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

SPARC Aggravates Blood-Brain Barrier Disruption via Integrin αVβ3/MAPKs/MMP-9 Signaling Pathway after Subarachnoid Hemorrhage.

Blood-brain barrier (BBB) disruption is a common and critical pathology following subarachnoid hemorrhage (SAH). We investigated the BBB disruption property of secreted protein acidic and rich in cysteine (SPARC) after SAH. A total of 197 rats underwent endovascular perforation to induce SAH or sham operation. Small interfering ribonucleic acid (siRNA) for SPARC or scrambled siRNA was administered intracerebroventricularly to rats 48 h before SAH. Anti-SPARC monoclonal antibody (mAb) 236 for functional blocking or normal mouse immunoglobulin G (IgG) was administered intracerebroventricularly 1 h after SAH. Selective integrin αVβ3 inhibitor cyclo(-RGDfK) or phosphate-buffered saline was administered intranasally 1 h before SAH, along with recombinant SPARC treatment. Neurobehavior, SAH severity, brain edema, immunohistochemical staining, and Western blot were evaluated. The expression of SPARC and integrin αVβ3 was upregulated after SAH in the endothelial cells. SPARC siRNA and anti-SPARC mAb 236 prevented neuroimpairments and brain edema through protection of BBB as measured by IgG extravasation 24 and 72 h after SAH. Recombinant SPARC aggravated neuroimpairments and cyclo(-RGDfK) suppressed the harmful neurological effects via inhibition of activated c-Jun N-terminal kinase, p38, and matrix metalloproteinase-9 followed by retention of endothelial junction proteins. SPARC may induce post-SAH BBB disruption via integrin αVβ3 signaling pathway.

Nano Diaminopropane tetrac and integrin αvβ3 expression in different cancer types: Anti-cancer efficacy and Safety

Integrins are a family of heterodimeric plasma membrane glycoproteins, which regulate tumor growth, angiogenesis, migration, and metastasis. Integrin αvβ3 has been recognized as a putative target for the treatment of several cancers. Thus, the characterization of αvβ3 distribution in different human tumors is of substantial interest in tumor targeting and its suppression. In this study we evaluated the expression of integrin αvβ3 in different cancer types to define the expression pattern in cancer model. Furthermore, we investigated the effect of novel αvβ3 antagonist Diaminopropane Tetraiodothyroacetic acid conjugated to poly (lactic-co-glycolic acid) polymer and its nanoformulated form (NDAT), on different cancer cell lines both in vitro and in xenografts. In vitro, NDAT downregulated αv and β3 monomer expression. In vivo in tumor xenografts, similarly, NDAT downregulated αv and β3. Distinct reduction in tumor weight and viability was observed in glioblastoma xenografts treated with NDAT. Furthermore, NDAT was safe and tolerable in mice treated with high doses. In conclusion, NDAT is an effective and safe inhibitor of integrin αvβ3 expression in various cancer types, which indicates its impact on the targetability and suppression of αvβ3-associated tumor functions.

Tropism of Coxsackie virus A9 depends on the +1 position of the RGD (arginine- glycine- aspartic acid) motif found at the C’ terminus of its VP1 capsid protein

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvβ6 and/or αvβ3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvβ3 and αvβ6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy.

Molecular design and anti-melanoma activity of a novel bullfrog antibacterial peptide RGD-chimera.

Melanoma is a common malignant skin tumor, which is the only fatal skin tumor at present. Melanoma has a high degree of malignancy and metastasis. The activity of modified Temporin-La (T-La) peptides from bullfrog skin were evaluated for antitumor activity and improved targeting in melanoma cells. The amino acid sequence of T-La was modified, resulting in the antitumor peptide, T-La (FS). T-La and T-La (FS) were coupled to the RGD small molecule polypeptide to form the chimeric peptides RGD-T-La and RGD-T-La (FS), respectively. The secondary structures for the peptides, evaluated using circular dichroism, were found to be α-helical. The structure of T-La was evaluated using bioinformatics. In addition, the antitumor effects of the modified peptide and the targeting of RGD chimeric peptide to the tumor in vivo and in vitro were analyzed. Antitumor activity was measured in vitro using the MTT assay. Tumor cells with high integrin αvβ3 expression were detected using flow cytometry, and tumor cells were screened for sensitivity to RGD-T-La (FS) to establish a tumor model in nude mice. The effects of the peptides on tumor cells were measured using laser confocal microscopy in real-time. The mechanism of the peptide antitumor activity in tumor cells was evaluated with scanning electron microscopy. B16 melanoma cells were the most sensitive to the peptides, for which the cell survival rate was 24.65% for 10 µg/ml RGD-T-La (FS). RGD-La (FS) had a rapid effect on tumor cells. RGD chimeric polypeptides exhibited site-targeting cytotoxic effects in tumor cells. In the B16 melanoma mouse model, the peptides exhibited antitumor effects against early melanoma development and induced tumor apoptosis, possibly by inhibiting VEGF and promoting caspase-3 expression. Overall, the present study provides a scientific basis for the application of small molecule antimicrobial peptides as targeted antitumor agents and lays the foundation for the clinical application of these peptides as antitumor drugs.

Downregulation of the αvβ6 Integrin via RGD Engagement Is Affinity and Time Dependent.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvβ6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFβ inhibition. High αvβ6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvβ6 affinity, slow dissociation) and SC-68448 (low αvβ6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvβ6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvβ6 cell surface repopulation was faster for SC-68448 compared with compound 1 In addition, αvβ6-dependent release of active TGFβ from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFβ was restored, whereas after washout of compound 1 the inhibition of TGFβ activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvβ6 in NHBE cells if present continuously. These observations suggest αvβ6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

Talin1 Regulates Endometrial Adhesive Capicity Through the Ras Signaling Pathway

Background: Blastocyst implantation is mainly depended on the adhesion between cells and cell matrix. Endometrial adhesion plays an important role in establishing embryo implantation, but the underlying mechanisms are remains unclear. Talin1 is a local adhesion complex protein that is necessary for cell adhesion and movement. However, the role and mechanisms of Talin1 in embryo implantation are still unclear. Methods: The expression of Talin1 and Integrin αvβ3 was measured in the receptive endometrium from the RIF (Recurrent implantation failure) cohort and NC (normal fertile control group) cohort. A JEG-3 trophoblast and endometrial epithelial cell adhesion model and pregnant mouse model were established. The molecular mechanism of Talin1-mediated cell adhesion was explored by RNA sequencing, RT-qPCR, as well as western blotting assays. Findings: Talin1 enhances endometrial cell adhesion by regulating the Ras signaling pathway, and ultimately facilitates embryo implantation. Interpretation: This study revealed the molecular mechanisms of regarding the pathogenesis of RIF caused by endometrial receptivity insufficiency. Further pharmacological research on the Ras signaling pathway would be valuable and might provide new therapeutic targets for RIF patients. Funding: This study was supported by grants from the National Natural Science Foundation of China and the Natural Science Foundation of Guangxi Zhuang Autonomous Region and the Guangxi Medical University Training Program for Distinguished Young Scholars Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: All participants provided written informed consent, and the study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. All animal experimental procedures were approved by the animal ethics committee of Guangxi Medical University in accordance with animal research guidelines.

Abnormally activated OPN/integrin \u03b1V\u03b23/FAK signalling is responsible for EGFR-TKI resistance in EGFR mutant non-small-cell lung cancer

BackgroundAcquired epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance limits the long-term clinical efficacy of tyrosine kinase-targeting drugs. Although most of the mechanisms of acquired EGFR-TKI resistance have been revealed, the mechanism of ~ 15% of cases has not yet been elucidated.MethodsCell viability was analysed using the Cell Counting Kit-8 (CCK-8) assay. Proteome profiler array analysis was performed to find proteins contributing to acquired EGFR-TKI resistance. Secreted OPN was detected by ELISA. Immunohistochemical analysis was conducted to detect expression of integrin αV in NSCLC tissue. The effect of VS-6063 on apoptosis and proliferation of PC9 gefitinib-resistant cells was detected by fluorescence-activated cell sorting (FACS) and clonogenic assays. A mouse xenograft model was used to assess the effect of VS-6063 on the sensitivity of PC9 gefitinib-resistant cells to gefitinib.ResultsOPN was overexpressed in acquired EGFR-TKI-resistant NSCLCs. Secreted OPN contributed to acquired EGFR-TKI resistance by activating the integrin αVβ3/FAK pathway. Inhibition of FAK signalling increased sensitivity to EGFR-TKIs in PC9 gefitinib-resistant cells both in vitro and in vivo.ConclusionsOPN contributes to acquired EGFR-TKI resistance by up-regulating expression of integrin αVβ3, which activates the downstream FAK/AKT and ERK signalling pathways to promote cell proliferation in NSCLC.

Integrin αvβ3-dependent thyroid hormone effects on tumour proliferation and vascularisation

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvβ3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvβ3, in two murine xenograft tumour models with and without integrin αvβ3 expression. Integrin αvβ3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvβ3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvβ3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvβ3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Virus Mimetic Shell-Sheddable Chitosan Micelles for siVEGF Delivery and FRET-Traceable Acid-Triggered Release.

Targeting vascular endothelial growth factor (VEGF) using small interfering RNA (siVEGF) has shown great potential in inhibiting the growth, proliferation, and migration of tumors by reducing the proliferation of blood vessels. On the basis of bionic principles, a novel pH-responsive and virus mimetic shell-sheddable chitosan (CS) micelles (CMs) as siRNA delivery system was introduced in this study. The cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) modified poly(enthylene glycol) (PEG) was conjugated to the HA2 modified chitosan via a hydrazone linkage (cRGD-PEG-Hz-CS-HA2). The cRGD-PEG-Hz-CS-HA2 conjugate could form micelles by interacting with the complex of octanal, Boc-l-lysine, and 9-d-arginine (9R) (octyl-Lys-9R) as a hydrophodic core forming agent, termed as cRGD-PEG-Hz-CS-HA2/octyl-Lys-9R (abbreviated as cRGD/HA2/Hz-CMs).The CMs modified with cRGD can accurately target glioma cells (U87MG cells) with high expression of αvβ3. The payloads of siVEGF were packed into the core of cRGD/HA2/Hz-CMs via electrostatic interaction and hydrophobic interaction. The intracellular cargo release was achieved by the pH-responsive lysis of the hydrazone bond in acidic environment of endosome. Moreover, the exposed HA2, as a pH-sensitive membrane-disruptive peptide, assists the escape of the carriers from endosome into cytosol. In addition, cRGD/HA2/Hz-CMs can effectively deliver siVEGF and silence VEGF gene expression in U87MG cells, leading to the significant tumor growth inhibition. This study demonstrates that cRGD/HA2/Hz-CMs can deliver and release siVEGF in a controlled manner, which was traced by the fluorescence resonance energy transfer (FRET) system in order to achieve RNAi-based anti-angiogenic treatment of cancer in vivo.

Abstract IA-13: Molecularly targeted imaging and treatment via the integrin αvβ6

Abstract The integrin αvβ6 is an epithelial-specific cell surface receptor that is generally undetectable in healthy adult epithelium but is significantly up-regulated in a wide range of epithelial derived cancers including almost 100% of pancreatic cancers. The low expression of αvβ6 in healthy tissue, the high αvβ6 expression in pancreatic cancer cells, and the correlation of expression with the metastatic phenotype of cancer support αvβ6 as a particularly attractive molecular target for both detection and treatment. We previously developed an αvβ6-targeted molecular imaging agent, 18F-αvβ6-binding-peptide (18F- αvβ6-BP), a peptide that has high affinity (nM) and selectivity for the integrin αvβ6 with favorable pharmacokinetics in tumor-bearing mice and non-human-primates, and translated this imaging agent into a first-in-human study in patients with breast, colon, lung and pancreatic cancer (NCT03164486/ IND #124336). The 18F-αvβ6-BP was well tolerated, with PET images demonstrating significant uptake of [18F]αvβ6-BP in both the primary lesion and metastases, including sub centimeter lesions in liver and lung. Building upon these data we have now developed the theranostics pair [68Ga]Ga αvβ6-BP (for detection) and [177Lu]Lu DOTA-ABM- αvβ6-BP (for treatment). Both constructs have demonstrated high affinity (low nM) and selectivity for the integrin αvβ6 both in vitro in cell binding assays and in vivo in mouse models, are rapidly internalized and are stable in both mouse and human serum. The [68Ga]Ga αvβ6-BP and [177Lu]Lu DOTA-ABM αvβ6-BP constructs showed favorable pharmacokinetics in pre-clinical studies, with predominantly renal excretion and good tumor: normal tissue ratios. The [177Lu]Lu DOTA-ABM αvβ6-BP was well tolerated in mice at over 50X the human equivalent dose. In a pre-clinical therapeutic efficacy study, mice in the control groups had met the end point by 69 days from start of treatment. In contrast, the mice in the treatment group had 100% survival at the end of the study i.e. at 120 days. The dosimetry values [177Lu]Lu DOTA-ABM αvβ6-BP extrapolated from the biodistribution data indicated the kidneys and the stomach are the dose limiting organs with the extrapolated values being significantly lower than the federal guidelines for human safety. In a single dose, 14-day GLP acute toxicology study with doses exceeding 100x the human equivalent dose no test article-related effects. We now propose a first-in-human pilot trial for the theranostic pair [68Ga]Ga αvβ6-BP and [177Lu]Lu DOTA-ABM αvβ6-BP in patients with locally advanced or metastatic pancreatic adenocarcinoma (PDAC). In this presentation I will described the design and development of both the diagnostic and the therapeutic constructs, their preclinical evaluation and describe the future plans for the first-in-human pilot trial for the theranostic pair. Citation Format: Julie L. Sutcliffe. Molecularly targeted imaging and treatment via the integrin αvβ6 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-13.

Lyso-Gb3 Increases αvβ3 Integrin Gene Expression in Cultured Human Podocytes in Fabry Nephropathy

Background: Podocyturia in Fabry nephropathy leads to glomerulosclerosis and kidney disease progression. Integrins are involved in podocyte attachment to the glomerular basement membrane. We hypothesized that in Fabry nephropathy, lyso-Gb3 could modulate αvβ3 expression in podocytes. Together with UPAR, the αvβ3 integrin is a key mechanism involved in podocyte detachment and podocyturia. Methods: In cultured human podocytes stimulated with lyso-Gb3, the mRNA expression of the ITGAV and ITGB3 genes encoding integrins αv and β3, respectively, was analyzed by RT-qPCR. Results: In cultured human podocytes, lyso-Gb3 at concentrations encountered in the serum of Fabry patients increased ITGAV and ITGB3 mRNA levels within 3 to 6 h. This pattern of gene expression is similar to that previously observed for PLAUR (UPAR) gene expression but is in contrast to the delayed (24 h) upregulation of other markers of podocyte stress and mediators of injury, such as CD80, TGFβ1, CD74, Notch1, and HES. Conclusions: Human podocyte stress in response to glycolipid overload in Fabry nephropathy, exemplified by lyso-Gb3, is characterized by an early increase in the expression of components of the αvβ3/UPAR system, which contrasts with the delayed rise in the expression of other mediators of podocyte injury. This suggests that the αvβ3/UPAR system may be a therapeutic target in Fabry nephropathy.

Dermal mesenchymal stem cells promoted adhesion and migration of endothelial cells by integrin in psoriasis

The unusual dilatation of dermal capillaries and angiogenesis played important roles in psoriasis. Some genes and proteins of dermal mesenchymal stem cells (DMSCs) from psoriasis are abnormal and related to the function of endothelial cells (ECs). The present study was aimed to evaluate whether psoriatic DMSCs could affect adhesion and migration of ECs through neovascularization-related integrins in psoriasis. Human DMSCs, collected from psoriasis lesions and healthy skin, respectively, were cocultured with human umbilical vein endothelial cells (HUVECs). The expression levels of three integrins, that is, αvβ3, αvβ5, and α5β1 in HUVECs were tested by quantitative real-time polymerase chain reaction and Western blot analysis. The adhesion and migration of HUVECs were detected by adhesion assay and migration assay. The results showed that in psoriasis group, the expression of αVβ3 and α5β1 of HUVECs markedly increased 2.50- and 3.71-fold in messenger RNA levels, and significantly increased 1.63- and 1.92-fold in protein levels, comparing to healthy control group (all p < .05). But β5 was not significantly different between the two groups (p > .05). In addition, compared with control, psoriatic DMSCs promoted HUVECs adhesion by 1.62-fold and migration by 2.91-fold (all p < .05). In conclusion, psoriatic DMSCs impact HUVECs adhesion and migration by upregulating the expression of integrins αVβ3 and α5β1.

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p&amp;lt;0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

Telomerase-specific oncolytic immunotherapy for promoting efficacy of PD-1 blockade in osteosarcoma.

Immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1) antibody have recently improved clinical outcome in certain cancer patients; however, osteosarcoma (OS) patients are refractory to PD-1 blockade. Oncolytic virotherapy has emerged as novel immunogenic therapy to augment antitumor immune response. We developed a telomerase-specific replication-competent oncolytic adenovirus OBP-502 that induces lytic cell death via binding to integrins. In this study, we assessed the combined effect of PD-1 blockade and OBP-502 in OS cells. The expression of coxsackie and adenovirus receptor (CAR), integrins αvβ3 and αvβ5, and programmed cell death ligand 1 (PD-L1) was analyzed in two murine OS cells (K7M2, NHOS). The cytopathic activity of OBP-502 in both cells was analyzed using the XTT assay. OBP-502-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). Subcutaneous tumor models for K7M2 and NHOS cells were used to evaluate the antitumor effect and number of tumor-infiltrating CD8+ cells in combination therapy. K7M2 and NHOS cells showed high expression of integrins αvβ3 and αvβ5, but not CAR. OBP-502 significantly suppressed the viability of both cells, in which PD-L1 expression and the release of ATP and HMGB1 were significantly increased. Intratumoral injection of OBP-502 significantly augmented the efficacy of PD-1 blockade on subcutaneous K2M2 and NHOS tumor models via enhancement of tumor-infiltrating CD8+ T cells. Our results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the efficacy of PD-1 blockade in OS.

Protective effects of quercetin on uterine receptivity markers and blastocyst implantation rate in diabetic pregnant mice

ObjectiveDiabetic women have different reproductive problems. In pregnant diabetic women, high rates of perinatal mortality, spontaneous abortion and congenital anomalies are observed. We hypothesized that quercetin, as an antidiabetic and phytoestrogen, might have protective effects on the embryo implantation in pregnant diabetic mice. We investigated the ameliorative effects of quercetin on the levels of serum estrogen and progesterone, rate of blastocyst implantation, and uterine receptivity markers in diabetic mice. Materials and methodsDiabetic and healthy female mice were treated with quercetin (30 mg/kg/day) four weeks before pregnancy. Plasma sex-steroid levels were determined on day 4 of pregnancy. Also, uteri were harvested for investigation of protein and mRNA expression changes. In another set of our study, implantation rate was determined on day 5 of pregnancy. ResultsOur results indicated that quercetin was significantly reduced blood glucose levels in diabetic mice. The number of implantation sites as well as serum estradiol level was reduced in diabetic mice, and then treatment with quercetin significantly increased both. On the other hand, insulin like growth factor1, integrin αvβ3, and cyclooxygenase2 mRNA expression in the uterus of diabetic mice were significantly reduced, and quercetin treatment augmented the expression level of these genes. Besides, the level of inactive β-catenin protein level in the uterus of diabetic mice was higher than normal group; treatment with quercetin reduced the level of inactive β-catenin protein as compared to diabetic mice. ConclusionWe conclude that administration of quercetin before pregnancy can probably alleviate reproductive problems in diabetic women likely via its estrogenic and antihyperglycemic effects.

Enhanced expression of αVβ3 integrin in villus and extravillous trophoblasts of placenta accreta.

Placenta accreta is one of the most serious complications in obstetrics and gynecology. Villous trophoblasts (VT) and extravillous trophoblasts (EVT) play a central role in normal placentation. Placenta accreta is characterized by abnormal invasion of EVT cells through the uterine layers, due to changes in several parameters, including adhesion proteins. Although αvβ3 integrin is a central adhesion molecule, participating in multiple invasive pathological conditions including cancer, data on placenta accreta are lacking. To study the expression pattern of αvβ3 integrin in placenta accreta in comparison with normal placentas. We collected tissue samples from placentas defined as percreta, the most severe presentation of placenta accreta and from normal control placentas (n = 10 each). The samples underwent protein extractions for analyses of αvβ3 expression by Western blots (WB) and a parallel tissue assessment by immunohistochemistry (IHC). WB results indicated significantly elevated αvβ3 integrin expression in the percreta samples compared to normal placentas. These elevated levels were mainly contributed by EVT cells, as demonstrated by IHC. αvβ3 integrin demonstrated a classical membranal expression in the VT cells, whereas a uniformly distributed expression was documented in the EVT cells. These patterns of the αvβ3 integrin localization were similar in both accreta and normal placental samples. Enhanced αvβ3 integrin expression, mainly in extra villous trophoblasts of placenta percreta, implies for a role of this adhesion molecule in pathological placentation.

Targeted Radionuclide Therapy in Patient-Derived Xenografts Using 177Lu-EB-RGD.

Currently, most patients with non-small cell lung cancer (NSCLC) are diagnosed in advanced stages with a poor five-year survival rate. Therefore, intensive research aimed at finding novel therapeutic strategies has been ongoing; experimental models that reliably emulate NSCLC disease are greatly needed to predict responses to novel therapeutics. Therefore, we developed patient-derived xenograft (PDX) models of NSCLC, which we then used to evaluate the therapeutic efficacy of 177Lu-EB-RGD, a peptide-based radiopharmaceutical with improved pharmacokinetics that targets integrin αvβ3 In this study, three different groups of NSCLC-PDXs were successfully established, all of which maintained the same IHC and genetic characteristics of the human primary tumor. The two NSCLC-PDX groups with intense and low expression of integrin αvβ3 (denoted as PDXαvβ3+ and PDXαvβ3-) were chosen as the experimental models to evaluate the in vivo biological behavior of 177Lu-EB-RGD. In SPECT imaging and biodistribution studies, 177Lu-EB-RGD showed significantly higher accumulation in PDXαvβ3+ and PDXαvβ3- models than its corresponding monomer 177Lu-RGD. A single dose of 18.5 MBq 177Lu-EB-RGD was enough to completely eradicate the tumors in PDXαvβ3+, with no sign of tumor recurrence during the observation period. Such treatment was also efficacious in PDXαvβ3-: a single dose of 29.6 MBq 177Lu-EB-RGD led to a significant delay in tumor growth as compared with that in the control or 177Lu-RGD group. The preclinical data from the use of this model suggest that 177Lu-EB-RGD may be an effective treatment option for NSCLC and should be further evaluated in human trials.