Cutaneous microvascular aging correlates with clinical disorders of the elderly

Many age-related changes in skin microvessels manifest clinically as familiar cutaneous disorders of the elderly. Purpura (^{Montagna and Carlisle 1979}), telangiectasia (^{Ryan, 1969}), palor (^{Tsuchida, 1993}), angioma, and venous lake formation (^{Havlik, 1999}) are among the more obvious examples of morphologic changes observed in aged skin. More subtle age-dependent changes in the physiologic function of skin microvessels have also been well documented and include decreased vasoreactivity (^{Algotsson et al, 1995}), altered inflammatory cell profiles (^{Ashcroft et al, 1998}), and impaired wound repair (^{Holt et al, 1992;Schafer et al, 1994}).

Pathophysiologic events associated with these clinical findings include significant age-dependent reductions in the total number of papillary loop microvessels, decreased thickness of microvessel basement membranes, and decreased numbers of perivascular cells (^{Braverman and Fonferko, 1982}). Thus, decreased perfusion and increased capillary fragility appear as obvious consequences of these structural alterations. Beyond these observations, however, it remains unclear as to what the major determinants of skin microvascular aging are or what their relative contribution to clinical signs and symptoms in aged skin might be.

Changes in the vascular system in general as a function of donor age, however, are associated with a number of functional and hemodynamic alterations commonly observed in cardiovascular disease states (^{Cooper et al, 1994}). The alterations point to a correlation between gradual loss of vascular cell homeostatic capacity and phenotypic alterations that characterize replicative senescence. Such alterations include changes in expression patterns of proteolytic activities (^{Millis et al, 1992;West et al, 1996;Ashcroft et al, 1997}), inflammatory markers (^{Maier et al, 1990,1993;Comi et al, 1995}), and cell structural proteins (^{Vasile et al, 2001}). Increased vasoconstrictor responses and decreases in both vasodilators and vasoprotective agents are known to associate with advancing age (^{Carrol and Schroff, 1991;Tokunaga et al, 1991;Sato et al, 1993;Cooper et al, 1994}). Age-dependent impairment of angiogenesis observed during tumor growth, ischemic vascular disease, and wound repair strongly suggest that the phenotypic and functional integrity of endothelial cells (EC) may be compromised in old versus young animals (^{Pili et al, 1994;Reed et al, 1998;Rivard et al, 1999}). Many of the in vivo manifestations of vascular aging are apparent in continuously passaged fibroblasts and EC in vitro (^{Cooper et al, 1994;Chang et al, 1997}), underscoring the relevance of using cell cultures derived from vascular tissues to examine the contribution of aging to vascular dysfunction.

Whether replicative senescence in EC plays a direct or indirect role in these observations remains unclear; however, survival mechanisms of EC are unique and closely related to cell fate decisions controlled by the cell cycle, DNA repair, and programmed cell death signaling pathways. It is not surprising to note that the patterns of gene expression observed in senescent EC are distinctly different from other cell types and many of these genes are related to cell survival pathways (^{Shelton et al, 1999;Vasile et al, 2001}).

An emerging theme in tissue repair processes that requires us to examine these pathways involves decreased proliferative capacity and activation of the senescent phenotype in aged vascular cells. The universality of replicative senescence remains uncertain however. For example, the recent demonstration of unlimited in vitro proliferation and lack of a senescence phenotype in certain cells of the rodent central nervous system (^{Mathon et al, 2001;Tang et al, 2001}) suggests that molecular triggers responsible for cell fate decisions are not identical in all tissues and across all species. Thus, clarification of mechanisms governing replicative senescence during periods of tissue stress and chronic disease states in humans specifically has important clinical implications.

Microvascular survival is an equilibrium between maintenance and remodeling

To achieve homeostasis, the rate of new blood vessel growth must equal the loss of old blood vessels due to vascular reorganization and apoptosis. EC play a major role in stabilizing and destabilizing vascular structures by balancing cellular processes of proliferation, quiescence, apoptosis, and senescence (Figure 1).

Figure 1.

Cellular proliferation, quiescence, apoptosis, and senescence are the four principal cytologic states that set the cutaneous microvascular in a dynamic balance between maintenance and remodeling. Actively growing and quiescent cells are the two reversible processes that would maintain a homeostasis between maintenance and vascular proliferation. Depending on the environmental conditions, EC in either quiescence or proliferation can move irreversibly into apoptosis (thus promoting remodeling) or into senescence. Cellular processes controlling EC survival are in a dynamic balance during remodeling and maintenance states of the microvasculature. Molecular mechanisms modulating EC proliferation and senescence must be balanced against competing processes of apoptosis and quiescence to maintain homeostasis and correct function during states of tissue stress (e.g., inflammation, wound repair, tumor growth, and environmental insults). According to theoretical notions on the contribution of cellular senescence to diseased states, EC that become senescent are regarded as having exited this homeostatic milieu and to have initiated a pathologic condition for the endothelium.

Full figure and legend (62K)

During angiogenesis, EC proliferation occurs in an area proximal to the tip of the new vessel and these structures represent sprouting postcapillary venules (^{Folkman and Bream, 1992}). Control of EC proliferation is considered a major therapeutic target in angiogenesis research and many different angiogenic and angiostatic factors are believed to act at the level of EC proliferation.

EC proliferation can be controlled by growth factor and extracellular matrix signaling pathways that are tightly coupled via matricellular proteins, such that endogenous angiostatic factors appear to sequester growth factors, block receptor activation, and even induce EC apoptosis (^{Bornstein, 1995;Sage, 1997;Kupprion et al, 1998;Lucas et al, 1998}). EC cell–cell signaling is an equally strong regulator of EC proliferation in vitro and signaling through intercellular contacts clearly regulates EC cell cycle machinery (^{Yoshizumi et al, 1995;Nakamura, 1997}).

The EC cell cycle can be arrested by three main mechanisms: (i) growth factor removal; (ii) extracellular matrix signaling a "nonpermissive" environment (^{Ingber, 1992}); and (iii) contact inhibition. The mechanisms of EC cell cycle arrest and induction of the quiescent state by the above three events are different but reversible. Many angiostatic factors work by G1 growth arrest and delay of entry into the S phase (^{Baldin et al, 1990;Funk, 1993;Abe et al, 1994;Gupta and Singh, 1994;Hori et al, 1994;Imamura et al, 1994}).

EC quiescence is not senescence as the latter represents an irreversible state of cell cycle arrest (see below). Near senescence, EC display several permanently activated processes such as increased pericellular proteolytic activity, a more disorganized extracellular matrix, an augmented inflammatory adhesion molecule profile, increased adhesion of monocytes, and altered cytoskeletal components controlling cell shape (^{Cooper et al, 1994;West, 1994;Chang et al, 1997;Vasile et al, 2001}). As these profiles can also be observed in aged skin and in aged vascular tissue, an exceptional parallelism between aging in vitro and in vivo is apparent.

Importantly, one should note that senescent-like phenotypes including critically shortened telomeres, can be achieved through chronic, external insults such as hyperoxia (^{von Zglinicki, 1995;Saretzki et al, 1998}). This suggests that environmental factors, as opposed to replicative exhaustion, can contribute to a senescent phenotype that leads in turn to dysfunctions in vascular homeostasis. Endothelial dysfunction in vivo occurs most prominently in aged vasculature and is characterized in part by increased superoxide production. Excess superoxide can trap nitric oxide (NO) into peroxynitrite derivatives, thus masking vasorelaxation responses in aged blood vessels (^{van Der Loo et al, 2000}). Such dysfunctional vasoreactivity involving NO expression is not restricted to aging vasculature and can be observed in the microvasculopathy of fibrotic disease states (^{Romero et al, 2000}). Persistent alterations in NO metabolism occur in EC derived from the skin of patients with scleroderma that appear to correlate with clinical symptoms (e.g., Raynaud's phenomenon). Interestingly, scleroderma-derived HDMEC also exhibit phenotypic and functional changes similar to senescent EC, such as decreased growth and a flattened cell morphology (^{Herron and Romero, 1998;Romero et al, 2000}). A better understanding of how hyperoxia and altered NO metabolism can mimic the effects of EC replicative senescence may lead to novel therapeutic interventions.

The adult endothelium in vivo is remarkably quiescent and EC divide very slowly unless activated in some way. Isolation and culture of EC result in a semiactivated state in which cells retain some specialized characteristics but lose others (^{Cines, 1998}). All human EC appear to retain the ability to divide many times in vitro but their continued survival depends on a variety of different factors and conditions (^{Bicknell, 1996}). When EC become activated via inflammatory cytokines, oxidative stress, or other pathologic insults many different vasoprotective factors are induced. Some of these include; NO (^{Dimmeler et al, 1997;Ceneviva et al, 1998}); Bcl-2 family members, A20 (^{Karsan et al, 1997}), MnSOD, 70 kDa HSP, heme oxygenase-1, angiopoietin-2, and VEGF (^{Ferrara and Davis-Smyth, 1997}). These vasoprotective agents can block induction of important cell fate decision components (e.g., p53, p21, p16, p27, Bax) while activating survival pathways (see below). Without the induction of such genes controlling vasoprotective factors, EC survival and replication would not match the loss of EC during states of inflammation. A state of chronic replication may, after a critical number of doublings, compromise the expression of these vasoprotective elements and thus the activation of the survival pathways.

The akt-nitric oxide signaling axis in endothelial cell survival

Recent evidence suggests the serine/threonine kinase Akt, also known as protein kinase B, plays a central role in modulating EC survival (^{Dimmeler and Zeiher, 2000}). Many different extracellular regulators of EC survival converge on the Akt signaling pathway via activation of phosphatidyl-inositol 3' kinase (PI3-K), as shown in Figure 2.

Figure 2.

Manifold EC surface-associated signaling events converge on a common survival kinase cascade system involving PI3-K and Akt. Activation via phosphorylation of Akt by PI3-K, in turn, differentially affects multiple other signaling pathways with downstream targets known to affect EC survival (e.g., endothelial NO synthetase, Raf, p65PAK1, and hTERT). Indirect blockade of apoptosis occurs via Akt-mediated phosphorylation of Bad, which does not bind bcl-2, allowing it to interact with bax and block caspase activation. p65PAK1 activation by other mechanisms directly affects phosphorylation of Bad, leading to the same result. A potential feedback loop(s) is apparent as telomerase-activation in EC leads to maintenance of activated Akt during induction of apoptosis (GSH, unpublished).

Full figure and legend (57K)

This cartoon depicts the activation of a series of signaling pathways by known EC survival factors: VEGF activation of KDR (^{Gerber et al, 1998}), flow-induced shear-stress (^{Dimmeler et al, 1998}), activation of Tie2 by Angiopoietin-2 (^{Suri, 1996;Tamura et al, 1999}), direct activation by the small G protein Ras (^{Rodriguez-Viciana et al, 1997}), as well as the inducement of the Gi-protein/PI3-kinase pathway via sphingosine-1-phosphate (^{Morales-Ruiz et al, 2001}). Activation of the PI3-K/Akt pathway in turn activates multiple other pathways that affect cell survival, including activation of p65/pAK1 (^{Dimmeler et al, 1999;Tang et al, 2001}) and phosphorylation of Raf leading to downstream inhibition of the Raf-MEK-ERK kinase cascade (^{Zimmermann and Moelling, 1999}). The activation of PI3-K and Akt leads to phosphorylation of Bad and the release of bcl-2. Free bcl-2 is then bound by bax and prevented from activating the caspase cascade, effectively blocking apoptosis.

Of particular importance to the mechanisms of vascular aging and survival is the recent observation that the Akt kinase is apparently involved in the phosphorylation of human telomerase reverse transcriptase subunit (hTERT) leading to telomerase activation (^{Kang et al, 1999;Breitschopf et al, 2001}). With phosphorylation, enzymatic activity of hTERT could be affected, which could, in turn, influence the resistance of EC to apoptosis (^{Yang et al, 1999}).

NO is a well-known vascular survival factor with pleomorphic effects on EC. NO possesses the intriguing property of inducing telomerase activity in early passage HUVEC, potentially leading to extended replicative capacity in EC (Vasa, 1999). This effect of NO qualifies it as not only a vasoprotective factor but also an antiaging agent that maintains a youthful vascular cell phenotype. NO may thus be involved in an hTERT-dependent feedback mechanism that is modulated by Akt kinase activity (see Figure 2). In fact, telomerized EC show a higher degree of eNOS induction, as well as higher basal levels of eNOS than their near senescent, parental counterparts. Consequently, telomerized EC respond better to shear stress than their parental, nontelomerized controls (Tsao, 2001). Stable transduction of hTERT in EC appears to induce a vasoprotective mechanism that is mediated, in part, through NO activity in EC.

Cell proliferation, death, and senescence are closely linked

Apoptosis and the cell cycle use parts of the same molecular machinery (^{King and Cidlowski, 1995;Meikarnatz and Schlegel, 1995;Kasten and Giordano, 1998}). It is well established that cells progressing through the cell cycle become more susceptible to apoptosis versus quiescent cells, but interestingly, cell cycle arrest in late G1 or S phase potentiates apoptosis. Cell cycle checkpoint proteins (e.g., p53, pRB, and cyclin-dependent kinase inhibitors, p21 and p27) are involved in making cell fate decisions of apoptosis or cycle arrest but precise mechanisms remain unclear (^{Evan and Littlewood, 1998}). It is known that unrepaired DNA and chromosomal damage triggers apoptotic induction, justifying these checkpoint proteins as "guardians of the genome" (^{Lane, 1992}).

As shown in Figure 3, chromosomal damage in the form of telomeric DNA shortening during cell division serves as a "biological clock" that triggers replicative senescence (^{Harley, 1992;Feng et al, 1995;Bodnar et al, 1998}).

Figure 3.

Telomeres cap the ends of chromosomes and represent specialized protein-nucleic acid structures with a unique DNA repeat sequence, TTAGGG. Mammalian DNA polymerase is unable to replicate near the ends of chromosomes and thus each round of cell division is associated with telomeric DNA shortening. The rate of shortening is unique in different tissues and cell types. Uncharacterized molecular sensing systems appear to monitor DNA damage at chromosomal ends such that at a critical length a gene expression program is activated, termed "cellular senescence", in which cells become unresponsive to growth signals and express the senescence phenotype.

Full figure and legend (72K)

Human telomeres represent tandom DNA repeats of the sequence TTAGGG/CCCTAA at the ends of chromosomes and these sequences are synthesized by a ribronucleoprotein complex called telomerase (^{Feng et al, 1995;Greider and Blackburn, 1996;Lingner et al, 1997;Lingner et al, 1997}). Telomerase is active in germline cells and progenitor stem-like cells but not in most human somatic tissues (^{Shay and Bacchetti, 1997}). Cell cycle arrest at senescence is a complex and as yet poorly defined process that involves genetic programming much like the differentiated phenotype (^{Sedivy, 1998}). There are many pathways that lead to the final common state of replicative senescence, but DNA damage is recognized as a major pathway involving p53-mediated G1 arrest (^{Di Leonardo et al, 1994}). The state of replicative senescence is considered an "activated" state by many investigators, particularly with regard to the expression of genes involved in extracellular matrix metabolism (^{West, 1994;Campisi et al, 1996}).

Ec survival and senescence pathways involve chromosomal ends

Like all somatic cells, human EC undergo replicative senescence after a finite number of divisions that varies between 20 and 50 population doublings (PD), depending on the tissue of origin and culture conditions. Aside from our current knowledge on telomere biology, we can only guess as to the critical factors involved in the senescence pathway of human EC. HUVEC appear to respond to autocrine production of IL-1 by undergoing senescence (^{Maier et al, 1990,1994}). VEGF was found to both delay the onset of replicative senescence in HDMEC and reverse "culture" senescence of HDMEC when added to cells grown without VEGF (^{Watanabe et al, 1997}). No changes in IL-1 could be detected in the latter study and telomerase activity was undetectable regardless of VEGF treatment (see below); however, senescent HDMEC expressed high levels of both p16 and p21 CDK inhibitors relative to VEGF-treated HDMEC and VEGF withdrawal increased p16 with little effect on p21. The data suggest that replicative senescence in HDMEC is associated with G1 growth arrest involving p16.

Replicative senescence may be bypassed by two theoretical cell cycle checkpoint "hurdles" (^{Shay et al, 1991}). The first hurdle termed the "Hayflick M1" point involves cell cycle arrest that can be overcome via transformation with viral oncogenes, SV40 LgTAg, Adenovirus E1A and HPV E6 and E7, and/or the inactivation of their cellular targets, pRB and p53. The second hurdle is the "M2 Crisis" point at which telomeric DNA shortening triggers massive genomic instability and eventually cellular death (^{Harley et al, 1992}). Rare mutations that activate telomerase activity and allow repair of telomeric DNA result in immortalization and the majority of human tumor cells express telomerase activity (^{Chiu and Harley, 1997}). Because cell cycle checkpoints have been bypassed, immortalized tumor cells do not possess a normal phenotype. This limits their utility as study material for life-span extension experiments.

Another way to avoid senescence is by ectopic expression of the hTERT gene in normal (pre-M1) cells (^{Bodnar et al, 1998}), and this imparts a replicative immortality in which cells maintain their functional characteristics and differentiated program without transformation to a neoplastic phenotype (^{Jiang et al, 1999;Morales et al, 1999}). An exception to the current view that hTERT alone confers replicative immortality on all cell types is the finding that both hTERT expression and inactivation of pRB or p16 is required to immortalize primary human keratinocytes and mammary epithelial cells (^{Kiyono et al, 1998}). It is now known, however, that "culture shock" defined as growth arrest due to the inability to adapt to tissue culture conditions in vitro is different from true replicative senescence accounting for the observations that telomerase activation alone is insufficient for cellular immortalization of all cell types (Woody Wright, personal communication). Nevertheless, there remains some uncertainty over the relative requirements for both hTERT and viral oncogene activation in the conditional immortalization of all somatic cell types including EC (^{O'Hare et al, 2001,Lagunoff et al, 2002;Venetsanakos et al, 2002;Krump-Kovalinkova et al, 2001;Arbiser et al, 2001}), underscoring the need for a comprehensive study on telomerase transduction in several different EC types for both in vitro and in vivo conditions.

Low telomerase activity has been found in several different types of EC; but this activity is rapidly lost during the first few divisions in vitro (^{Hsiao et al, 1997;Yang et al, 1999}). Re-activation of endogenous EC telomerase can be accomplished in vitro by ectopic expression of oncogenes, such as HPV E6 and E7 (^{Rhim et al, 1998}), but such oncogenic gene expressions have both pleiotrophic and transformative effects. It is therefore apparent that HDMEC must possess all components of the telomerase holoenzyme (RNA, regulatory components) to express telomerase activity hTERT; however, the hTERT component, which is not expressed, is the rate-limiting factor. Similar findings were found in other cell types (^{Bodnar et al, 1998;Jiang et al, 1999}).

Ectopic hTERT expression can reactivate telomerase in HDMEC and the resultant "telomerized" cell lines appear to maintain the phenotypic and functional characteristics of young primary HDMEC for many cell divisions (^{Yang et al 1999}). Because telomere dynamics and telomerase activity have been linked to apoptosis (^{Kondo et al, 1998;Fu et al, 1999;Hahn et al, 1999;Holt et al, 1999;Karlseder et al, 1999}), however, and because telomerized HDMEC exhibit resistance to apoptotic induction relative to in vitro aged EC (^{Yang et al, 1999}), the manner in which telomerase activation affects HDMEC programmed cell death pathways warrants further investigation.

Implications of telomere-telomerase survival systems in cutaneous vascular biology

Whereas the majority of research on skin cell telomeres and telomerase is currently focused on epithelial regeneration and neoplasia (^{Taylor et al, 1996;Ueda et al, 1997;Sakabe et al, 1998;Kunimura et al, 1998}), it is clear that wound repair responses of both the epidermal and the dermal compartments are dependent on the critical length of telomeric DNA (^{Rudolph et al, 1999}). Little is known about exactly which cell type(s) of the dermis depend on restoration of depleted telomere length by telomerase for normal repair processes or how telomere dynamics participate in cutaneous aging and pathology. Dermal EC are likely to activate hTERT during proliferative responses (e.g., wound healing, inflammation) and possibly tumor angiogenesis, but no studies have demonstrated a change in EC telomere length associated with disease states.¹ It is known that dermal explants from human venous stasis ulcers display shortened replicative lifespan and a hypertophic phenotype consistent with senescent cells (^{Mendez, 1998;Mendez et al, 1999;Raffetto et al, 1999}). Likewise, in states of oxidative tissue stress observed during the vasospastic stages of scleroderma, dermal EC express a senescent phenotype and altered expression of NO (^{Romero et al, 2000}). As vascular repair systems depend on the proliferative responses of EC and as aging and chronic pathology of large blood vessels are associated with intimal telomeric erosion (^{Chang and Harley, 1995}), it is likely that telomere loss plays a significant role in skin vascular survival mechanisms in vivo.

Three key discoveries and development of new model systems help to clarify potential mechanisms by which telomerase activity may participate in cutaneous vascular homeostatic and stress responses: (i) the existence of bone marrow-derived vascular stem cells; (ii) DNA damage-induced telomere length sensing systems in human (post natal) somatic cells; and (iii) the creation of animal model systems to study telomere biology.

Only recently has evidence emerged that circulating EC (angioblastic) precursors may be recruited to sites of vascular remodeling in humans, and EC survival factors (e.g., VEGF) appear to play important roles in this process (^{Isner and Asahara, 1999}). Because it is known that stem and germline cells are among the only cells in postnatal tissues that continue to express telomerase activity (^{Kolquist et al, 1998}), it is possible that cutaneous microvasculature harbors angioblastic cells under conditions of tissue stress and these cells help to repopulate depleted EC populations. Characterization of telomerase expression and other markers of amplifying cells in subpopulations of dermal perivascular cells may help to clarify the regenerative capacity of skin microvasculature.

Conversely, telomerase repression in adult somatic cells appears to be responsible for the lack of telomere length maintenance in postnatal tissues (^{Bodnar et al, 1998}). Factors that modulate telomerase activity and/or telomere dynamics in somatic cells are the current focus of intensive research efforts (^{Evans, 2000;McEachern et al, 2000;Colgin et al, 2000; White et al, 2000; Stampfer et al, 2001;Beattie et al, 2001}). The interrelationships between various EC survival pathways, apoptosis, and telomere biology holds the promise of a better understanding of these processes and provides a rationale for therapeutic intervention during states of chronic skin pathologies in which the proliferative capacity of cells is compromised.

Animal models of tumor blood vessel growth demonstrate that host age is a strong determinant of both angiogenic potential and tumor growth in vivo (^{Pili et al, 1994;Rivard et al, 1999}). Two new mouse models have been developed that could be used to examine the effects of aging, telomere dynamics, and EC survival systems on microvascular remodeling in vivo. One utilizes telomerized human EC transplanted into SCID mice (^{Yang et al, 2001}), whereas the other employs the use of the telomerase RNA component knockout mouse (mTR–/–) (^{Rudolph et al, 1999}). In the former model system, fully functional human capillaries that form anastamoses with host murine vessels can be tracked in vivo using eGFP-labeled, telomerized HDMEC (Figure 4).

Figure 4.

Visualization of human microvasculature created by xenografting telomerized HDMEC in SCID mice. Primary HDMEC were transduced with both eGFP and hTERT, FAC-sorted to purify fluorescent cells, and then subcutaneously implanted in SCID mice. After 2 wk, grafts were harvested and analyzed by IF microscopy. Fluorescent vascular structures prove the human origin of the microvessels and allow visualization of vessel morphology. Such a system represents an in vivo human cutaneous microvascular remodeling platform for analysis of EC survival mechanisms, wound repair, and vascular cell gene delivery.

Full figure and legend (174K)

We found telomerized HDMEC formed microvessels that exhibited superior durability with time after grafting versus vessels created with in vitro-aged primary HDMEC (^{Yang et al, 2001}), and thus we speculate that direct correlations exist between telomerase activity, telomere length, and angiogenic potential in vivo.

In late generations of the mTR–/– mouse, decreased repair responses were noted after skin wounding (^{Rudolph et al, 1999}). Although no reports have appeared directly demonstrating that telomere length and/or telomerase activity affect host angiogenesis in the mTR–/– mouse, it is well known that microvascular remodeling is a critical component of wound repair and thus it is possible that decreased angiogenic potential represents a key factor that contributes to these observations.² Both the mTR–/– mouse and the SCID-HDMEC models may therefore be very useful for studying the effect of telomeric erosion and telomerase activity as important determinants of EC differentiated function in vivo.

Recent advances in tissue engineered skin substitutes and grafting technologies have advanced the field of cutaneous wound repair (^{Phillips, 1998;Singer and Clark, 1999}), but incorporation of vascular components remains an elusive goal. Slowing the rate of telomere erosion via activation of endogenous telomerase or ectopic expression of hTERT could have profound clinical implications in organ and tissue transplantation strategies (^{Shay and Wright, 2000}), and thus formation of human telomerized microvasculature in vivo represents an important development (^{Yang et al, 2001}). We hope that the contribution of EC precursors to skin repair processes and pathologic states can be studied by utilizing such in vivo model systems. Similarly, clarification of telomere-dependent and -independent signaling pathways influencing EC aging and survival represent key areas of research that may be approached utilizing dermal microvascular EC assay systems.

Notes

¹ While this manuscript was in press, Osanai et al, showed that dermal EC activity telomerase during the healing of human skin (Osanai, personal communication)

² While this manuscript was in press,^{Franco et al, 2002}, showed that angiogenesis was decreased in the MTR–/– mouse both in the Matrigel plug assay and in response to B16 melanoma implantation.

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Acknowledgments

This work was supported in part by NIH PO-1 AR44012, the Carl J. Herzog Foundation, Geron Corporation, and the Dermatology Foundation. J. Yang received a Research Fellowship from Warner Lambert Consumer Healthcare through the Dermatology Foundation. GSH is a Terman Fellow. We thank Choy-Pik Chiu, Calvin B. Harley, and Jane Lebkowski for their invaluable scientific and editorial insights.