Abstract-Cardiac fibroblasts (CFs) produce extracellular matrix proteins and participate in the remodeling of the heart.It is unknown if brain natriuretic peptide (BNP) is synthesized by CFs and if BNP participates in the regulation of extracellular matrix turnover. In this study, we examined the production of BNP in adult canine CFs and the role of BNP and its signaling system on collagen synthesis and on the activation of matrix metalloproteinases (MMPs Key Words: cardiac fibroblasts Ⅲ extracellular matrix Ⅲ remodeling Ⅲ cGMP Ⅲ protein kinase G T he cardiac interstitium is a dynamic structure, as reflected by continuous synthesis and degradation of matrix proteins. The family of matrix metalloproteinases (MMPs) consists of more than 20 different zinc-containing, Ca 2ϩ -dependent endopeptidases. 1,2 They degrade matrix proteins and therefore play an important role in the physiological regulation of the interstitium. The interstitial collagenases (MMP-1 and MMP-13), the stromelysin (MMP-3), the gelatinases (MMP-2 and MMP-9), and membranous-type 1 MMPs (MMP-14; MT1-MMP) have been demonstrated within the mammalian myocardium. 2 Furthermore, dysregulation of MMP proteins and their endogenous inhibitor, tissue inhibitors of MMP (TIMP), has been observed in the hypertensive and the failing heart, suggesting an important role of MMP in the process of ventricular remodeling. [3][4][5][6][7] Cardiac fibroblasts (CFs) play a crucial role in the regulation of the extracellular matrix (ECM) of the heart by synthesizing collagen and other matrix proteins as well as promoting their degradation by secreting MMP proteins. In response to myocardial injury, activation of CFs occurs. These activated CFs (myofibroblasts) have special morphological and functional characteristics. 8,9 The natriuretic peptides (NPs) atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) play important roles in maintaining cardiorenal homeostasis under physiological and pathological conditions. 10 ANP and BNP are synthesized by cardiomyocytes, and their production is stimulated in pathologic conditions such as myocardial infarction (MI), cardiac hypertrophy, and heart failure (HF). 11-13 ANP and BNP have natriuretic, vasodilating, and lusitropic properties, and they inhibit the sympathetic and renin-angiotensin-aldosterone system. 14,15 These actions are primarily mediated by the second messenger cGMP. 16 Cameron et al 17 have recently reported that ANP is produced in CFs after MI, indicating that fibroblasts, like cardiomyocytes, can be a source of NPs. However, it remains unknown if BNP is produced by CFs.Although it is well established that BNP has growthinhibiting properties in the heart, 18 -21 the role of BNP on the regulation of the cardiac interstitium remains undefined. Given the widespread cross-talk of the NPs with other systems that are activated in cardiorenal disorders, we aimed to investigate whether CFs are a source of BNP and whether BNP and its signaling system contribute to the regulation of Original

Mammalian cells have developed diverse strategies to restrict retroviral infection. Retroviruses have therefore evolved to counteract such restriction factors, in order to colonize their hosts. Tripartite motif-containing 5 isoform-alpha (TRIM5alpha) protein from rhesus monkey (TRIM5alpharh) restricts human immunodeficiency virus type 1 (HIV-1) infection at a postentry, preintegration stage in the viral life cycle, by recognizing the incoming capsid and promoting its premature disassembly. TRIM5alpha comprises an RBCC (RING, B-box 2 and coiled-coil motifs) domain and a B30.2(SPRY) domain. Sequences in the B30.2(SPRY) domain dictate the potency and specificity of the restriction. As TRIM5alpharh targets incoming mature HIV-1 capsid, but not precursor Gag, it was assumed that TRIM5alpharh did not affect HIV-1 production. Here we provide evidence that TRIM5alpharh, but not its human ortholog (TRIM5alphahu), blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. The specificity for this restriction is determined by sequences in the RBCC domain. Our observations suggest that TRIM5alpharh interacts with HIV-1 Gag during or before Gag assembly through a mechanism distinct from the well-characterized postentry restriction. This finding demonstrates a cellular factor blocking HIV-1 production by actively degrading a viral protein. Further understanding of this previously unknown restriction mechanism may reveal new targets for future anti-HIV-1 therapy.

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNalpha treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNalpha was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNalpha in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNalpha and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNalpha-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNalpha-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin (FN). FN contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the natriuretic peptide receptor A (NPR-A) sequence contains both RGD and DDX sequences. The goal of the current investigation was to determine potential interactions between FN and NPR-A on BNP induction of cGMP in cultured human cardiac fibroblasts (CFs). Further, we sought to determine whether a Mayo designed NPR-A specific RGD peptide could modify this interaction. Here we reconfirm the presence of all three natriuretic peptide receptors (NPR) in CFs. CFs plated on FN demonstrated a pronounced increase in cGMP production to BNP compared to non-coated plates. This production was also enhanced by the NPR-A specific RGD peptide, which further augmented FN associated cGMP production. Addition of HS-142-1, a NPR-A/B antagonist, abrogated the responses of BNP to both FN and the NPR-A specific RGD peptide. Finally, we defined a possible role for the NPR-C through non-cGMP mechanisms in mediating the anti-proliferative actions of BNP in CFs where the NPR-C antagonist cANF 4-28 but not HS-142-1 blocked BNP-mediated inhibition of proliferation of CFs. We conclude that NPR-A interacts with components of the ECM such as FN to enhance BNP activation of cGMP and that a small NPR-A specific RGD peptide augments this action of BNP with possible therapeutic implications. Lastly, the NPR-C may also have a role in mediating anti-proliferative actions of BNP in CFs.

Murine primary cells are poorly permissive to human immunodeficiency virus type 1 (HIV-1) vector infection. Retroviral infectivity is influenced by dominant inhibitors such as TRIM5␣. Sensitivity to TRIM5␣ is altered by interactions between cyclophilin A and the HIV-1 capsid. Here we demonstrate that competitive inhibitors of cyclophilins, cyclosporine or the related Debio-025, stimulate HIV-1 vector transduction of primary murine cells, including bone marrow and macrophages, up to 20-fold. Unexpectedly, the infectivity of an HIV-1 mutant or a simian lentivirus that does not recruit cyclophilin A is also stimulated by these drugs. We propose that cyclosporine and related compounds will be useful tools for experimental infection of murine primary cells. It is possible that HIV-1 infection of murine cells is inhibited by dominant factors related to immunophilins.