Inhibition of KLF5-Myo9b-RhoA Pathway-Mediated Podosome Formation in Macrophages Ameliorates Abdominal Aortic Aneurysm

RATIONALE: Abdominal aortic aneurysms (AAAs) are characterized by pathological remodeling of the aortic wall. Although both increased Krüppel-like factor 5 (KLF5) expression and macrophage infiltration have been implicated in vascular remodeling, the role of KLF5 in macrophage infiltration and AAA formation remains unclear. OBJECTIVE: To determine the role of KLF5 in AAA formation and macrophage infiltration into AAAs. METHODS AND RESULTS: KLF5 expression was significantly increased in human AAA tissues and in 2 mouse models of experimental AAA. Moreover, in myeloid-specific Klf5 knockout mice (myeKlf5-/- mice), macrophage infiltration, medial smooth muscle cell loss, elastin degradation, and AAA formation were markedly decreased. In cell migration and time-lapse imaging analyses, the migration of murine myeKlf5-/- macrophages was impaired, and in luciferase reporter assays, KLF5 activated Myo9b (myosin IXB) transcription by direct binding to the Myo9b promoter. In subsequent coimmunostaining studies, Myo9b was colocalized with filamentous actin, cortactin, vinculin, and Tks5 in the podosomes of phorbol 12,13-dibutyrate-treated macrophages, indicating that Myo9b participates in podosome formation. Gain- and loss-of-function experiments showed that KLF5 promoted podosome formation in macrophages by upregulating Myo9b expression. Furthermore, RhoA-GTP levels increased after KLF5 knockdown in macrophages, suggesting that KLF5 lies upstream of RhoA signaling. Finally, Myo9b expression was increased in human AAA tissues, located in macrophages, and positively correlated with AAA size. CONCLUSIONS: These data are the first to indicate that KLF5-dependent regulation of Myo9b/RhoA is required for podosome formation and macrophage migration during AAA formation, warranting consideration of the KLF5-Myo9b-RhoA pathway as a therapeutic target for AAA treatment

T cell reconstitution in patients after cell therapy.

<p>P1, a child with Fanconi anaemia, underwent a second mismatched donor, CD34 selected stem cell graft after in the context of relapsed MDS. Donor HSVTK/CD34 modified T cells were infused in two dose aliquots and were detectable at low level in peripheral blood for over 12 weeks before the patient died of disease relapse. The persistence of non-modified T cells reflects the reduced intensity conditioning and absence of serotherapy. P2, an infant with RAG1 deficient SCID had no pre-existing T cell immunity and was conditioned whist infected with H1N1 influenza. Modified T cells persisted for over 12 months, with eventual recovery of thymic derived donor T cells after one year and normalisation of immunity. P3 suffered Ligase IV deficiency, a form of radiosensitive SCID. Expansion of modified donor T cells was detected within two weeks of first infusion, but the patient died from mucositis related pulmonary and gastrointestinal haemorrhage before dose escalation.</p

GMP T cell transduction reagents.

<p>GMP T cell transduction reagents.</p

Transfer and tracking of T cell mediated virus specific immunity.

<p>Most compelling, and beneficial, was transfer of immunity against pandemic H1N1 infulenza in P2. The haploidentical donor had been electively vaccinated against the strain before leukapheresis harvest of peripheral blood lymphocytes. The transduced and CD34 enriched populations exhibited specific IFNγ responses against HI1N1 compared to non-stimulated control cells. Samples collected 150 days after donor lymphocyte infusion from the patient showed similar H1N1 specific IFNγ responses, which coincided with clearance of persistent H1N1 respiratory infection. These responses were still detectable after 350 days.</p

Production of donor HSVTK-CD34 T cells.

<p>Production of donor HSVTK-CD34 T cells.</p

GMP compliant T cell transduction procedure.

<p>GMP compliant T cell transduction procedure.</p

T cell repertoire diversity before and after modification.

<p>Complementarity determining region-3 (CDR3) T-cell receptor (TCR) spectratyping was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077106#pone.0077106-Qasim3" target="_blank">[18]</a>. Briefly, RNA was extracted and cDNA prepared from pre- and post-transduced cells. Twenty four Vβ-specific primers were used with a fluorescent-labelled constant region (Cβ)-specific primer to RT-PCR amplify the CDR3 region of the TCR β chain. Products were run on an AB3130 Genetic Analyzer and analysed using GeneMapper v4.0 software (Applied Biosystems, Warrington, UK). Representative data for P2 is showing preservation Vβ family distributions is shown.</p

Vector configuration and study schema.

<p>1a. A gamma retroviral platform incorporating long terminal repeals (LTRs) from Myeloproliferative sarcoma virus (MPSV) and leader sequence 71 derived from Murine embryonic stem cell virus (MESV). The splice site corrected herpes simplex virus thymidine kinase suicide gene (scHSVTK) fused to a truncated (splice variant) human CD34 gene is shown. 1b. Subjects undergoing CD34 selected mismatched allografts and receiving grafts carrying <5×10⁴ T cells/kg following conditioning (but not serotherapy) were eligible. Gene modified T cells were scheduled at two cell doses, the first 5×10⁴/kg the day following the stem cell graft, and the second programmed within 28 days at a higher dose of 5×10⁵/kg. In the event of GVHD>Grade I, Ganciclovir therapy was scheduled for seven days to eliminate gene modified T cells.</p

Transduction, enrichment and suicide gene function.

<p>(a) Flow cytometry of peripheral blood lymphocytes after transduction. Cells were activated with anti-CD3/28 beads and underwent two rounds of exposure to vector before removal of activation beads and magnetic bead enrichment using a CliniMacs device. (b) Transduced T cells were enriched (CD34+) to >90% purity for all three products. (c) Upon exposure to the prodrug Ganciclovir (GCV, 10 uM), engineered cells from all three donors had reduced survival compared to non-modified controls (P<0.001). Means of triplicate wells and standard error of means are shown.</p