We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. Several approaches are being developed to image reporter gene expression in living animals. These include methods that rely on charge-coupled device camera imaging and bioluminescent reporter genes (1), single-photon emission computed tomography using the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene (2), approaches that use magnetic resonance imaging (reviewed in ref.3), methods based on the HSV1-tk reporter gene and positron emission tomography (PET) (4, 5), and the use of the dopamine type 2 receptor (D2R) as a reporter gene for PET (6). The use of reporter genes that can be imaged in vivo will permit many different applications, including monitoring of both somatic gene transfer and transgenic͞knock-in reporter gene expression (7).PET provides repeated, noninvasive imaging of biological processes in living subjects (8, 9). PET utilizes molecular probes labeled with positron-emitting radioisotopes (e.g., fluorine-18, with a half-life of 110 min). PET probes typically are either positron-labeled ligands for receptors or positron-labeled substrates for intracellular enzymes. Tracer quantities of PET probes yield a tomographic image after their retention, as a consequence of either binding of positron-labeled ligand to a receptor or conversion of positron-labeled substrate to ''trapped'' metabolic product(s). PET is particularly well suited for application to human studies. PET reporter gene imaging in humans will allow monitoring of the location(s), magnitude, and duration of therapeutic͞suicide gene expression, by using vectors for DNA delivery in which the reporter gene and therapeutic gene are expressed from a common transcript (10,11 HSV1-tk refers to the gene, HSV1-TK refers to the enzyme.) HSV1-TK phosphorylates a range of substrates, including acycloguanosines (e.g., acyclovir, ganciclovir, penciclovir) and uracil derivatives [e.g., 2Ј-f luoro-2Ј-deoxy-1-␤-arabinofuranosyl-5-iodouracil (FIAU)]. In contrast, mammalian thymidine kinases phosphorylate acycloguanosines only minimally, making these substrates advantageous as reporter gene imaging probes (11). Acycloguanosine derivatives are currently used extensively both as cytotoxic pharmaceuticals to treat herpes infections and for HSV1-tk suicide gene therapy (13).Improvements in sensitivity of the HSV1-tk reporter gene imaging assay can be achieved either (i) by identifying substrates that exhibit higher V max ͞K m for HSV1-TK or (ii) by engineering TK enzyme(s) with improved V max ͞K m for a particular reporter substrate. Decreased V max ͞K m of HSV1-TK for thymidine (an endogenous competitor) also should improve HSV1-tk reporter gene assay sensiti...

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8

TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.

Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS proteinprotein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6 -22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor L-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/ BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH. Pulmonary arterial hypertension (PAH)2 is a devastating disease, characterized by increasing pulmonary arterial pressures, leading to right heart failure and eventually, death (1, 2). Recent studies demonstrate an important role for BMPRII in the pathogenesis of the disease when mutations to the BMPR2 gene were identified as a major underlying cause of heritable or familial PAH (FPAH) (3-5). Approximately 144 different mutations in 210 patients with FPAH have now been identified (6 -8). Additionally, it is estimated that 20% of patients with idiopathic PAH (IPAH) harbor mutations to the BMPR2 gene. These mutations may be a previously undetected inherited mutation or arise from sporadic mutation of the gene.BMPRII is widely expressed in normal tissues. In the lung vasculature, BMPRII is most highly expressed on endothelial cells (9) and at lower levels in smooth muscle cells and fibroblasts. Expression of BMPRII is markedly reduced in the pulmonary vasculature of patients with FPAH and IPAH, regardless of whether they harbor mutations in the BMPR2 gene (9).Pulmonary artery smooth muscle cells (PASMCs) from patients with mutations in BMPRII have a reduced capacity to activate Smad1/5. This is coupled with a reduced ability of BMPs to inhibit proliferation of PASMCs (10). In contrast, the BMPRII ligands BMP2 and BMP4 promote proliferation, migration, and survival in PAECs (11, 12). The opposite effects of BMPs on PAECs and PASMCs provide a convincing model for pulmonary vascular damage in PAH, where a loss of BMP...