Redefine photoprotection: Sun protection beyond sunburn.

Excessive exposure to ultraviolet (UV) light leads to acute and chronic UV damage and is the main risk factor for the development of skin cancer. In most countries with western lifestyle, the topical application of sunscreens on UV-exposed skin areas is by far the most frequently used preventive measure against sunburn. Further than preventing sunburns, increasing numbers of consumers are appreciating sunscreens with a medium- to high-level sun protective factor (SPF) as basis for sustainable-skin ageing or skin cancer prevention programs. However, recent investigations indicate that clinically significant DNA damages as well as a lasting impairment of cutaneous immunosurveillance already occur far below the standard of one minimal erythema dose (MED) sunburn level, which contributes to the current discussion of the clinical value of high-protective SPF values. Ex vivo investigations on human skin showed that the application of SPF30 reduces DNA damage for a day long sun exposure (24 MED) drastically by about 53% but is significantly surpassed by SPF100 reducing DNA damage by approx. 73%. Further analysis on different SPF protection levels in UV-exposed cell culture assays focusing on IL-18, cell vitality and cis/trans-urocanic acid support these findings. Whereas SPF30 and SPF50+ sunscreens already offer a solid UVB cover for most indications, our results indicate that SPF100 provides significant additional protection against mutagenic (non-apoptotic-) DNA damage and functional impairment of the cutaneous immunosurveillance and therefore qualifies as an optimized sunscreen for specifically vulnerable patient groups such as immunosuppressed patients, or skin cancer patients.

Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility.

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.

New approach to prevent myocardial hypertrophy: the import blocking peptide.

Calcineurin, a serine/threonine phosphatase, plays a crucial role in the development of myocardial hypertrophy. Calcineurin is a cytosolic phosphatase that dephosphorylates the nuclear factor of activated T cells (NFAT), a transcription factor. Until now, it has been postulated that dephosphorylated NFAT is shuttled into the nucleus. Recent evidence demonstrates that not only NFAT, but also calcineurin, is localized in the nucleus. Once calcineurin and NFAT enter the nucleus of cardiomyocytes, transcription of genes that are characteristic for myocardial hypertrophy (e.g., brain natriuretic peptide and atrial natriuretic peptide) occurs. Although the exact nuclear function of calcineurin remains unclear, its co-existence with NFAT is important for the full transcriptional activity of the calcineurin/NFAT signaling cascade. The principal effect of nuclear calcineurin is likely the prolonged nuclear retention period of NFAT. Potential effects of nuclear calcineurin include an antagonistic function to glycogen synthase kinase 3beta, which phosphorylates NFAT for its export out of the nucleus, or direct antagonization of the export of NFAT, catalyzed by the chromosome region maintenance 1, which would leave NFAT nuclear. The nuclear localization sequence (NLS) region at the amino acid sequence from position 172 to 183 of calcineurin Abeta is essential for shuttling calcineurin into the nucleus by importinbeta(1). A synthetic import blocking peptide (IBP) that mimics the nuclear localization sequence of calcineurin was generated. The NLS analog on IBP saturates the calcineurin binding site of importinbeta(1). This prevents the binding of calcineurin to importin and inhibits the nuclear shuttling of calcineurin. Inhibition of the calcineurin/importinbeta(1) interaction by competing synthetic peptides represents a new approach to the inhibition of the development of myocardial hypertrophy.

Inhibition of nuclear import of calcineurin prevents myocardial hypertrophy.

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinbeta(1) to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [(3)H]-leucine incorporation (159%+/-9 versus 111%+/-11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%+/-11 versus 133%+/-8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%+/-9 versus 270%+/-11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.

Fibroblast migration after myocardial infarction is regulated by transient SPARC expression.

Secreted protein, acidic, and rich in cysteine (SPARC) is thought to regulate cell matrix interaction during wound repair. We hypothesized that SPARC might promote migration via integrin-dependent mechanisms. The present study was designed to clarify the contribution of SPARC in the wound healing process after myocardial infarction (MI). Adult mice received a specific alpha(v) integrin inhibitor or vehicle through osmotic mini pumps. Mice of each group were either sham-operated or MI was induced. SPARC expression was investigated 2 days, 7 days, and 1 month after the surgical procedure. For migration assays, a modified Boyden chamber assay was used. A transient increase of SPARC levels was observed, starting at day 2 (2.55+/-0.21), day 7 (3.72+/-0.28), and 1 month (1.9+/-0.16) after MI. After 2 months, SPARC expression dropped back to normal levels compared to sham-operated hearts. Immunofluorescence analysis showed an increase of SPARC in the infarcted area 2 days after MI, a strong increase in the scar area 7 days after MI, and only low levels in the scar area 2 months after MI. Integrin alpha(v) inhibition abolished the up-regulation of SPARC. In vitro migration assays demonstrated that fibronectin-stimulated haptotaxis of fibroblasts was modulated by SPARC. This study provides evidence that SPARC is significantly up-regulated in the infarcted region after MI. This up-regulation is dependent on alpha(v) integrins. As SPARC is found to regulate fibroblast migration, it appears to play an important role in the injured myocardium with regard to healing and scar formation.