Cardiac function and architecture are maintained in a model of cardiorestricted overexpression of the prorenin-renin receptor.

The (pro)renin-renin receptor, (P)RR has been claimed to be a novel element of the renin-angiotensin system (RAS). The function of (P)RR has been widely studied in renal and vascular pathology but the cardio-specific function of (P)RR has not been studied in detail. We therefore generated a transgenic mouse (Tg) with cardio-restricted (P)RR overexpression driven by the alpha-MHC promotor. The mRNA expression of (P)RR was ∼ 170-fold higher (P<0.001) and protein expression ∼ 5-fold higher (P<0.001) in hearts of Tg mice as compared to non-transgenic (wild type, Wt) littermates. This level of overexpression was not associated with spontaneous cardiac morphological or functional abnormalities in Tg mice. To assess whether (P)RR could play a role in cardiac hypertrophy, we infused ISO for 28 days, but this caused an equal degree of cardiac hypertrophy and fibrosis in Wt and Tg mice. In addition, ischemia-reperfusion injury was performed in Langendorff perfused isolated mouse hearts. We did not observe differences in parameters of cardiac function or damage between Wt and Tg mouse hearts under these conditions. Finally, we explored whether the hypoxia sensing response would be modulated by (P)RR using HeLa cells with and without (P)RR overexpression. We did not establish any effect of (P)RR on expression of genes associated with the hypoxic response. These results demonstrate that cardio-specific overexpression of (P)RR does not provoke phenotypical differences in the heart, and does not affect the hearts' response to stress and injury. It is concluded that increased myocardial (P)RR expression is unlikely to have a major role in pathological cardiac remodeling.

Activation of liver X receptor-alpha reduces activation of the renal and cardiac renin-angiotensin-aldosterone system.

Liver X receptor (LXR)-alpha is a pivotal player in reverse cholesterol metabolism. Recently, LXR-alpha was implicated as an immediate regulator of renin expression in a cAMP-responsive manner. To determine whether long-term LXR-alpha activation affects activation of the renal and cardiac renin-angiotensin-aldosterone system (RAAS), we treated mice with T0901317 (T09, a specific synthetic LXR agonist) in combination with the RAAS inducer isoproterenol (ISO). LXR-alpha-deficient (LXR-alpha(-/-)) and wild-type (WT) C57Bl/6J mice were treated with ISO, T09 or both for 7 days. Low-dose ISO treatment, not associated with an increase in blood pressure, caused an increase in renal renin mRNA, renin protein and ACE protein in WT mice. WT mice treated with both ISO and T09 had decreased renal renin, ACE and AT(1)R mRNA expression compared with mice treated with ISO only. Cardiac ACE mRNA expression was also reduced in the hearts of WT mice treated with ISO and T09 compared with those treated with ISO alone. The transcriptional changes of renin, ACE and AT(1)R were mostly absent in mice deficient for LXR-alpha, suggesting that these effects are importantly conferred through LXR-alpha. In conclusion, LXR-alpha activation blunts ISO-induced increases in mRNA expression of renin, AT(1)R and ACE in the heart and kidney. These findings suggest a role for LXR-alpha in RAAS regulation.

Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosis-inducing ligand fusion protein with specificity for human CD7.

Current treatment of human T-cell leukemia and lymphoma is predominantly limited to conventional cytotoxic therapy and is associated with limited therapeutic response and significant morbidity. Therefore, more potent and leukemia-specific therapies with favorable toxicity profiles are urgently needed. Here, we report on the construction of a novel therapeutic fusion protein, scFvCD7:sTRAIL, designed to induce target antigen-restricted apoptosis in human T-cell tumors. ScFvCD7:sTRAIL consists of the death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7. Treatment with scFvCD7:sTRAIL induced potent CD7-restricted apoptosis in a series of malignant T-cell lines, whereas normal resting leukocytes, activated T cells, and vascular endothelial cells (human umbilical vein endothelial cells) showed no detectable apoptosis. The apoptosis-inducing activity of scFvCD7:sTRAIL was stronger than that of the immunotoxin scFvCD7:ETA. In mixed culture experiments with CD7-positive and CD7-negative tumor cells, scFvCD7:sTRAIL induced very potent bystander apoptosis of CD7-negative tumor cells. In vitro treatment of blood cells freshly derived from T-acute lymphoblastic leukemia patients resulted in marked apoptosis of the malignant T cells that was strongly augmented by vincristin. In conclusion, scFvCD7:sTRAIL is a novel recombinant protein causing restricted apoptosis in human leukemic T cells with low toxicity for normal human blood and endothelial cells.

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR.

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Exceptionally potent anti-tumor bystander activity of an scFv:sTRAIL fusion protein with specificity for EGP2 toward target antigen-negative tumor cells.

Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells). The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-to-bystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication) between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.

Coronaviruses as vectors: position dependence of foreign gene expression.

Coronaviruses are the enveloped, positive-stranded RNA viruses with the largest RNA genomes known. Several features make these viruses attractive as vaccine and therapeutic vectors: (i) deletion of their nonessential genes is strongly attenuating; (ii) the genetic space thus created allows insertion of foreign information; and (iii) their tropism can be modified by manipulation of the viral spike. We studied here their ability to serve as expression vectors by inserting two different foreign genes and evaluating systematically the genomic position dependence of their expression, using a murine coronavirus as a model. Renilla and firefly luciferase expression cassettes, each provided with viral transcription regulatory sequences (TRSs), were inserted at several genomic positions, both independently in different viruses and combined within one viral genome. Recombinant viruses were generated by using a convenient method based on targeted recombination and host cell switching. In all cases high expression levels of the foreign genes were observed without severe effects on viral replication in vitro. The expression of the inserted gene appeared to be dependent on its genomic position, as well as on the identity of the gene. Expression levels increased when the luciferase gene was inserted closer to the 3' end of the genome. The foreign gene insertions generally reduced the expression of upstream viral genes. The results are consistent with coronavirus transcription models in which the transcription from upstream TRSs is attenuated by downstream TRSs. Altogether, our observations clearly demonstrate the potential of coronaviruses as (multivalent) expression vectors.