Faculty Bibliography

BACKGROUND AND OBJECTIVES/OBJECTIVE:Interstitial photodynamic therapy (PDT) is an emerging modality for the treatment of solid organ disease. Our group at the University of Pennsylvania has performed extensive studies that demonstrate the feasibility of interstitial PDT for prostate cancer. Our preclinical and clinical experience is herein detailed. STUDY DESIGN/MATERIALS AND METHODS/METHODS:We have treated 16 canines in preclinical studies, and 16 human subjects in a Phase I study, using motexafin lutetium-mediated PDT for recurrent prostate adenocarcinoma. Dosimetry of light fluence, drug level and oxygen distribution for these patients were performed. RESULTS:We demonstrate the safe and comprehensive treatment of the prostate using PDT. However, there is significant variability in the dose distribution and the subsequent tissue necrosis throughout the prostate. CONCLUSIONS:PDT is an attractive option for the treatment of prostate adenocarcinoma. However, the observed variation in PDT dose distribution translates into uncertain therapeutic reproducibility. Our future focus will be on the development of an integrated system that is able to both detect and compensate for dose variations in real-time, in order to deliver a consistent overall PDT dose distribution.

Members of the myocardin-related family of transcription factors play critical roles in regulating vascular smooth muscle and cardiac differentiation. To examine the function of myocardin-related transcription factor (MRTF)-B, mice were generated from ES cells harboring a conditional insertional mutation, or gene trap, of the MRTF-B gene. Expression of the MRTF-B mutant allele results in a fusion protein consisting of the N terminus of MRTF-B fused to beta-galactosidase, which is functionally null. Homozygous MRTF-B gene trap mice (MRTF-B-/-) die between embryonic day (E)17.5 and postnatal day 1 from cardiac outflow tract defects. MRTF-B is expressed in the premigratory neural crest, in rhombomeres 3 and 5, and in the neural crest-derived mesenchyme surrounding the aortic arch arteries. Consistent with the pattern of expression, E10.5 and E11.5 MRTF-B-/- mutants exhibit deformation of aortic arch arteries 3, 4, and 6 and severe attenuation of smooth muscle cell differentiation in the arch arteries and the aorticopulmonary septum, despite normal migration and initial patterning of cardiac neural crest cells. Remarkably, the observed pathology was rescued and viable mice generated by intercrossing MRTF-B mutants with mice expressing Cre recombinase under the transcriptional control of the neural crest-restricted Wnt-1 promoter, which results in restoration of normal MRTF-B expression in the neural crest. Taken together, these studies reveal that MRTF-B plays a critical role in regulating differentiation of cardiac neural crest cells into smooth muscle and demonstrate that neural crest-derived smooth muscle differentiation is specifically required for normal cardiovascular morphogenesis.

The SAP domain transcription factor myocardin plays a critical role in the transcriptional program regulating smooth muscle cell differentiation. In this report, we describe the capacity of myocardin to physically associate with megakaryoblastic leukemia factor-1 (MKL1) and characterize the function of MKL1 in smooth muscle cells (SMCs). The MKL1 gene is expressed in most human tissues and myocardin and MKL are co-expressed in SMCs. MKL1 and myocardin physically associate via conserved leucine zipper domains. Overexpression of MKL1 transactivates serum response factor (SRF)-dependent SMC-restricted transcriptional regulatory elements including the SM22alpha promoter, smooth muscle myosin heavy chain promoter/enhancer, and SM-alpha-actin promoter/enhancer in non-SMCs. Moreover, forced expression of MKL1 and SRF in undifferentiated SRF(-/-) embryonic stem cells activates multiple endogenous SMC-restricted genes at levels equivalent to, or exceeding, myocardin. Forced expression of a dominant-negative MKL1 mutant reduces myocardin-induced activation of the SMC-specific SM22alpha promoter. In NIH3T3 fibroblasts MKL1 localizes to the cytoplasm and translocates to the nucleus in response to serum stimulation, actin treadmilling, and RhoA signaling. In contrast, in SMCs MKL1 is observed exclusively in the nucleus regardless of serum conditions or RhoA signaling. However, when actin polymerization is disrupted MKL1 translocates from the nucleus to the cytoplasm in SMCs. Together, these data were consistent with a model wherein MKL1 transduces signals from the cytoskeleton to the nucleus in SMCs and regulates SRF-dependent SMC differentiation autonomously or in concert with myocardin.

The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF(-/-) ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22 alpha promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22 alpha, smooth muscle alpha-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.

Cytoskeletal proteins play important roles in regulating cellular morphology, cytokinesis and intracellular signaling. In this report, we describe a developmentally regulated gene encoding a novel cell lineage-restricted cytoskeletal protein, designated SM22beta. SM22beta shares high-grade sequence identity with the smooth muscle cell (SMC)-specific protein, SM22alpha, the neuron-specific protein, NP25, and the Drosophila melanogaster flight muscle-specific protein, mp20. The mouse SM22beta cDNA encodes a 199-amino acid polypeptide that contains a single conserved calponin-like repeat domain. During mouse embryonic development, the SM22beta gene is expressed in a temporally and spatially regulated pattern in the tunica media of arteries and veins, endocardium and compact layer of the myocardium, bronchial epithelium and mesenchyme of the lung, gastrointestinal epithelium and cartilaginous primordia. During postnatal development, SM22beta is co-expressed with SM22alpha in arterial and venous SMCs. In addition, SM22beta is expressed at high levels in the bronchial epithelium and lung mesenchyme, gastrointestinal epithelial cells and in the cartilagenous and periosteal layer of bones. Three-dimensional deconvolution microscopic analyses of A7r5 SMCs revealed that SM22beta co-localizes with SM22alpha to cytoskeletal actin filaments. Taken together, these data demonstrate that SM22beta is a novel actin-associated protein with a unique cell lineage-restricted pattern of expression.

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22alpha promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5'-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.