Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration.

OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.

Protein kinase Cα (PKCα) regulates bone architecture and osteoblast activity.

Bones' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca(-/-) female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca(-/-) but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca(-/-) mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca(-/-) mice do not. Female Prkca(-/-) mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca(-/-) mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions.

A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications.

Adenoviruses (Ads) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector "ChAdOx1" in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the coxsackie and adenovirus receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of an RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumor-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

A targeted single mutation in influenza A virus universal epitope transforms immunogenicity and protective immunity via CD4+ T cell activation.

CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.

Impact of obesity and SARS-CoV-2 infection: implications for host defence - a living review.

The role of obesity in the pathophysiology of respiratory virus infections has become particularly apparent during the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, where obese patients are twice as likely to suffer from severe coronavirus disease 2019 (COVID-19) than healthy weight individuals. Obesity results in disruption of systemic lipid metabolism promoting a state of chronic low-grade inflammation. However, it remains unclear how these underlying metabolic and cellular processes promote severe SARS-CoV-2 infection. Emerging data in SARS-CoV-2 and Influenza A virus (IAV) infections show that viruses can further subvert the host's altered lipid metabolism and exploit obesity-induced alterations in immune cell metabolism and function to promote chronic inflammation and viral propagation. In this review, we outline the systemic metabolic and immune alterations underlying obesity and discuss how these baseline alterations impact the immune response and disease pathophysiology. A better understanding of the immunometabolic landscape of obese patients may aid better therapies and future vaccine design.

Finasteride metabolism and pharmacogenetics: new approaches to personalized prevention of prostate cancer.

Incidences of prostate cancer in most countries are increasing owing to better detection methods; however, prevention with the use of finasteride, a very effective steroid 5α-reductase type II inhibitor, has been met with mixed success. A wide interindividual variation in response exists and is thought to be due to heritable factors. This article summarizes the literature that attempts to elucidate the molecular mechanisms of finasteride in terms of its metabolism, excretion and interaction with endogenous steroid molecules. We describe previously reported genetic variations of steroid-metabolizing genes and their potential association with finasteride efficacy. Based on the literature, we outline directions of research that may contribute to understanding the interindividual variation in finasteride prevention and to the future development of personalized medicine.

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications.

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

Cancer Antigen Discovery Is Enabled by RNA Sequencing of Highly Purified Malignant and Nonmalignant Cells.

PURPOSE: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. EXPERIMENTAL DESIGN: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested. RESULTS: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer. CONCLUSIONS: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

CD4+ T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features.

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.

Histone deacetylase inhibitor trichostatin A sensitises cisplatin-resistant ovarian cancer cells to oncolytic adenovirus.

Ovarian cancer is often termed a silent killer due to the late onset of symptoms. Whilst patients initially respond to chemotherapy, they rapidly develop chemo-resistance. Oncolytic adenoviruses (OAds) are promising anti-cancer agents engineered to "hijack" the unique molecular machinery of cancer cells enabling tumour-selective viral replication. This allows spread to adjacent cells and amplification of oncolysis within the tumour. OAds represent an excellent opportunity for ovarian cancer therapy via intra-peritoneal delivery, however the efficacy of OAds thus far is limited. Here, we evaluate chromatin (histone) modification in chemo-resistant cells and its relationship to Ad efficacy (wild-type or oncolytic Ad). In contrast to cisplatin-sensitive A2780 cells that show an efficient reduction of cell viability by Ad in the presence of cisplatin, cisplatin-resistant A2780/cp70 cells show diminishing Ad-mediated reduction of cell viability with escalating doses of cisplatin. Histone deacetylase (HDAC)-2 and to a lesser extent HDAC1 were up-regulated in cisplatin-resistant but not cisplatin-sensitive cells. Cisplatin-resistant cells treated with a pan-HDAC inhibitor trichostatin A (TsA) significantly enhanced Ad-mediated reduction of cell viability in the presence of cisplatin. Cells treated with TsA alone did not reduce cell viability suggesting these findings are Ad-dependent. Thus, we identify HDAC inhibition as a potential means to sensitise cisplatin-resistant ovarian cancer cells to virotherapies, an observation that may offer improved outcomes for patients with late stage, chemotherapy-resistant ovarian cancer.

Targeting Wnt/ß-Catenin Activated Cells with Dominant-Negative N-cadherin to Reduce Neointima Formation.

Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/ß-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/ß-catenin/TCF pathway. Infection with Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis (3 ± 0.2% versus 9 ± 0.7%; p < 0.05, n = 6) and significantly inhibited VSMC migration by 83 ± 15% (p < 0.05, n = 6), but did not affect VSMC proliferation (p > 0.05, n = 5). In an ex vivo human saphenous vein organ culture model, luminal delivery of Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis after 7 days of culture (4 ± 1.4% versus 9 ± 1.6%; p < 0.01, n = 6) and suppressed intimal thickening by 75 ± 7% (p < 0.05, n = 5), without a detrimental effect on endothelial cell coverage. In vivo, Ad-TOP-dn-N-cadherin significantly reduced intimal thickening at day 21 (n = 10) in comparison to the Ad-ß-galactosidase (Ad-ß-gal) control virus (n = 12, p < 0.05) in the mouse carotid artery ligation model. In summary, we have developed a novel approach to selectively reduce intimal thickening, which may be beneficial in reducing late vein graft failure.

Pseudotyped αvß6 integrin-targeted adenovirus vectors for ovarian cancer therapies.

Encouraging results from recent clinical trials are revitalizing the field of oncolytic virotherapies. Human adenovirus type 5 (HAdV-C5/Ad5) is a common vector for its ease of manipulation, high production titers and capacity to transduce multiple cell types. However, effective clinical applications are hindered by poor tumor-selectivity and vector neutralization. We generated Ad5/kn48 by pseudotyping Ad5 with the fiber knob domain from the less seroprevalent HAdV-D48 (Ad48). The vector was shown to utilize coxsackie and adenovirus receptor (CAR) but not CD46 for cell entry. A 20-amino acid peptide NAVPNLRGDLQVLAQKVART (A20) was inserted into the Ad5. Luc HI loop (Ad5.HI.A20) and Ad5/kn48 DG loop (Ad5/kn48.DG.A20) to target a prognostic cancer cell marker, αvß6 integrin. Relative to the Ad5.Luc parent vector, Ad5.HI.A20, Ad5.KO1.HI.A20 (KO1, ablated CAR-binding) and Ad5/kn48.DG.A20 showed ~ 160-, 270- and 180-fold increased transduction in BT-20 breast carcinoma cells (αvß6high). Primary human epithelial ovarian cancer (EOC) cultures derived from clinical ascites provided a useful ex vivo model for intraperitoneal virotherapy. Ad5.HI.A20, Ad5.KO1.HI.A20 and Ad5/kn48.DG.A20 transduction was ~ 70-, 60- and 16-fold increased relative to Ad5.Luc in EOC cells (αvß6high), respectively. A20 vectors transduced EOC cells at up to ~ 950-fold higher efficiency in the presence of neutralizing ovarian ascites, as compared to Ad5.Luc. Efficient transduction and enhanced cancer-selectivity via a non-native αvß6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvß6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases.

Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications.

Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies.