WRN mutations in Werner syndrome patients: genomic rearrangements, unusual intronic mutations and ethnic-specific alterations.

Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere.

Senescence-induced alterations of laminin chain expression modulate tumorigenicity of prostate cancer cells.

Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs) are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM alpha4 and beta2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM alpha4 or beta2 chain or both chains. Increased expression of either the LM alpha4 or beta2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer.

Nitric oxide effects on the function of aged cells ex vivo and in vivo.

BACKGROUND: Angiogenesis is impaired in most aged tissues. Accordingly, there is great interest in interventions that improve the ability of aged cells to undergo blood vessel formation and subsequent tissue repair. MATERIALS AND METHODS: Nitric oxide (NO), a mediator proposed to enhance angiogenesis, was administered (as the precursor SNAP, S-nitroso-N-acetylpenicillamine) to aortic ring explants from aged mice and to aged mice in two separate in vivo experiments: a PVA sponge implant model of angiogenesis and full thickness excisional dermal wounds. RESULTS: SNAP inhibited angiogenesis from the mouse aortic ring explants. However, there was a trend toward increased blood vessel formation in the sponges from the aged mice treated with SNAP. SNAP did not detectably enhance dermal wound healing or angiogenesis, but it significantly inhibited epidermal closure. CONCLUSION: These data underscore the complexity of using a single agent, even one with multiple mechanisms such as NO, to improve a clinical outcome such as angiogenesis or wound repair in aged animals.

Endothelial precursor cells.

The discovery and subsequent characterization of endothelial precursor cells (EPCs) has stimulated interest in their potential use in older persons. Understanding the mechanisms that underlie EPC availability and function has important clinical implications for this age group. In this review, we discuss aspects of EPCs that are relevant to their role in angiogenesis and cardiovascular disease. We then review the limited data on features of EPCs that are known to be altered in aging and might better define their clinical utility in older persons.

Culture of murine aortic explants in 3-dimensional extracellular matrix: a novel, miniaturized assay of angiogenesis in vitro.

Assays of angiogenesis in vitro are critical to the study of vascular morphogenesis and to the evaluation of therapeutic compounds that promote or inhibit vascular growth. Culture of explanted aortic segments from rats or mice in a 3-dimensional extracellular matrix (ECM) is one of the most effective ways to generate capillary-like endothelial sprouts in vitro. We have modified the classic aortic explant model by placing the aortic segments from mice within small (5.6 mm diameter, 30 microl volume) lenticular hydrogels of type I collagen supported at the edge by nylon mesh rings. This method of culture, referred to as the "miniature ring-supported gel" (MRSG) assay, optimizes handling, cytological staining, and conventional imaging of the specimen and permits use of minimal volumes of reagents in a 96-well tissue culture format. We have used the MRSG assay to quantify the impaired angiogenic response of aged mice relative to young mice and to show that aged mice have significantly decreased sprout formation, but have similar levels of invasion of vascular smooth muscle cells into the supportive ECM. The MRSG assay, which combines low volume, physically robust gels in conjunction with mouse aortic segments, may prove to be a highly useful tool in studies of the process and control of vascular growth.

The effects of aging on tumor growth and angiogenesis are tumor-cell dependent.

It is generally accepted that histologically similar tumors grow more slowly, with less angiogenesis, in aged mice relative to young mice. We subcutaneously implanted TRAMP-C2 tumor cells, a prostate cancer cell line not previously examined in aging, into syngeneic C57/Bl6 young (4 month) and aged (20 month) mice and compared tumor growth and angiogenesis. Unexpectedly, the prostate tumors grew as fast in aged as in young mice. Angiogenesis in TRAMP-C2 tumors was robust, with no differences between the young and aged mice in the number of vessels, distribution of vessel sizes or features of vessel maturation. Aged mice had lower levels of serum testosterone than the young mice. VEGF levels were similar in the tumors and sera of the young and aged mice. Comparison with B16/F10 melanoma, a cancer cell line that is representative of previous studies in aged mice, showed that B16/F10 tumors grew minimally in the aged mice. In contrast to the B16/F10, TRAMP-C2 tumors had an extracellular matrix with significantly higher levels of MMP2 and MMP9 expression and activity. These unique results demonstrate that tumor progression can be as robust in aged tissues as young tissues. The ability of aged mice to grow large, vascularized prostate tumors is associated with high levels of MMP2/9 activity that may produce a permissive environment for tumor growth and angiogenesis. These data demonstrate that tumor-cell specific features determine the effect of aging on tumor growth and angiogenesis.

Age-related differences in repair of dermal wounds and myocardial infarcts attenuate during the later stages of healing.

BACKGROUND: It is unclear whether delays in wound repair due to the age of the host persist into the later stages of healing. Late stage healing of dermal wounds and myocardial infarcts in rodents was examined to determine if aged animals "catch up" to their younger counterparts. MATERIALS AND METHODS: Excisional dermal wounds (5 mm) were made by punch biopsy and myocardial infarctions were produced by ligation of the left anterior descending coronary artery in young and aged mice and rats, respectively. Dermal wounds at day 11 and myocardial infarctions at day 14 were analyzed for wound area, angiogenesis, deposition of basement membrane proteins, and remodeling of collagen. RESULTS: Analyses demonstrated that wound areas, the deposition of basement membrane proteins and angiogenic responses were similar in young and aged rodents at late stages of wound repair. The dermal wounds of young mice had larger quantities of mature, compacted collagen fibers relative to aged mice, but immature collagen fibers predominated in myocardial infarcts in both young and aged rats. CONCLUSION: These results show that, with the exception of dermal collagen remodeling, aged animals catch up to their young counterparts with respect to many features of tissue repair. Consequently, therapies that target age-related deficiencies in healing will be most effective when administered shortly after the initial insult.