Transgenic expression of 15-lipoxygenase 2 (15-LOX2) in mouse prostate leads to hyperplasia and cell senescence.

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate, and is often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we generated prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks arachidonic acid-metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67(+) cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem or progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia or carcinoma and, mechanistically, prostate lobes (especially those of 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-betagal, p27(Kip1) and heterochromatin protein 1gamma staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.

Sequential segmental neuromuscular stimulation reduces fatigue and improves perfusion in dynamic graciloplasty.

Dynamic graciloplasty is used as a treatment modality for total urinary incontinence caused by a paralyzed sphincter. A problem with this application is undesirable fatigue of the muscle caused by continuous electrical stimulation. Therefore, the neosphincter must be trained via a rigorous regimen to transform it from a fatigue-prone state to a fatigue-resistant state. To avoid or shorten this training period, the application of sequential segmental neuromuscular stimulation (SSNS) was examined. This form of stimulation proved previously to be highly effective in acutely reducing fatigue caused by electrical stimulation. The contractile function and perfusion of gracilis muscles employed as neosphincters were compared between conventional, single-channel, continuous stimulation, and multichannel sequential stimulation in 8 dogs. The sequentially stimulated neosphincter proved to have an endurance 2.9 times longer (as measured by halftime to fatigue) than continuous stimulation and a better blood perfusion during stimulation (both of which were significant changes, p < 0.05). Clinically, this will not antiquate training of the muscle, but SSNS could reduce the need for long and rigorous training protocols, making dynamic graciloplasty more attractive as a method of treating urinary or fecal incontinence.

Sequential segmental neuromuscular stimulation: an effective approach to enhance fatigue resistance.

Electrical stimulation of skeletal muscle flaps is used clinically in applications that require contraction of muscle and force generation at the recipient site, for example, to assist a failing myocardium (cardiomyoplasty) or to reestablish urinary or fecal continence as a neo-sphincter (dynamic graciloplasty). A major problem in these applications (muscle fatigue) results from the nonphysiologic manner in which most of the fibers within the muscle are recruited in a single burst-like contraction. To circumvent this problem, current protocols call for the muscle to be put through a rigorous training regimen to transform it from a fatigue-prone to a fatigue-resistant state. This process takes several weeks during which, aside from becoming fatigue-resistant, the muscle loses power and contraction speed. This study tested the feasibility of electrically stimulating a muscle flap in a more physiologic way; namely, by stimulating different anatomical parts of the muscle sequentially rather than the entire muscle all at once. Sequential segmental neuromuscular stimulation (SSNS) allows parts of the muscle to rest while other parts are contracting. In a paired designed study in dogs (n = 7), the effects of SSNS on muscle fatigability and muscle blood perfusion in gracilis muscles were compared with conventional stimulation: SSNS on one side and whole muscle stimulation on the other. In SSNS, electrodes were implanted in the muscles in such a way that four separate segments of each muscle could be stimulated separately. Then, each segment was stimulated so that part of the muscle was always contracted while part was always resting. This type of stimulation permitted sequential yet continuous force generation. Muscles in both groups maintained an equal amount of continuous force. In SSNS muscles, separate segments were stimulated so that the duty cycle for any one segment was 25, 50, 75, or 100 percent, thus varying the amount of work and rest that any segment experienced at any one time. With duty cycles of 25, 50, and 75 percent, SSNS produced significantly (p < 0.01) enhanced resistance to fatigue. In addition, muscle perfusion was significantly (p < 0.01) increased in these sequentially stimulated muscles compared with the controls receiving whole muscle stimulation. It was concluded that SSNS reduces muscle fatigue and enhances muscle blood flow during stimulation. These findings suggest that using SSNS in clinical myoplasty procedures could obviate the need for prolonged training protocols and minimize problems associated with muscle training.

Three parameters optimizing closed-loop control in sequential segmental neuromuscular stimulation.

In conventional dynamic myoplasties, the force generation is poorly controlled. This causes unnecessary fatigue of the transposed/transplanted electrically stimulated muscles and causes damage to the involved tissues. We introduced sequential segmental neuromuscular stimulation (SSNS) to reduce muscle fatigue by allowing part of the muscle to rest periodically while the other parts work. Despite this improvement, we hypothesize that fatigue could be further reduced in some applications of dynamic myoplasty if the muscles were made to contract according to need. The first necessary step is to gain appropriate control over the contractile activity of the dynamic myoplasty. Therefore, closed-loop control was tested on a sequentially stimulated neosphincter to strive for the best possible control over the amount of generated pressure. A selection of parameters was validated for optimizing control. We concluded that the frequency of corrections, the threshold for corrections, and the transition time are meaningful parameters in the controlling algorithm of the closed-loop control in a sequentially stimulated myoplasty.

A novel in vitro system to study extravasated tumor cell-induced angiogenesis.

Angiogenesis, the formation of new blood vessels from preexisting ones, is a fundamental stage in the metastatic pathway. For the primary tumor, this neovascularization provides nutrients and oxygen as well as a route by which metastatic tumor cells gain access to the circulatory system. Among these metastatic tumor cells, there are subgroups of cells that express an angiogenesis-inducing cells phenotype (AICs) as well as others that do not. Tumor cells not expressing the angiogenesis-inducing cells phenotype (non-AICs) invade new tissues and remain as dormant micrometastases unless they accompany AICs. Thus, either alone or with non-AICs, angiogenesis-inducing cells form rapidly growing, clinically detectable metastases. Much of the current research in this area is concentrated on the vascularization of primary tumors, but the regulation of angiogenesis by extravasating or invading tumor cells has not being extensively studied. We have developed a working model, which demonstrates that human metastatic prostate cancer cells (PC-3) appear to induce human vascular endothelial cells (HUVECs) to translocate across a Matrigel-coated 8 mm membrane. The parameters of this model (i.e. pore size, seeding-cell density, seeding times) were established using highly invasive murine melanoma cells (B16F10) seeded on murine microvascular endothelial cells (CD3). We have further modified our model in order to include a host compartment made of collagen gel, in order to mimic the in vivo site of metastases-induced angiogenesis.