Multimodal approach in assessment of the response of breast cancer to neoadjuvant chemotherapy.

The ability for noninvasive visualization of functional changes of a tumor's oxygenation and circulatory system offers new advantages for prognosis and monitoring of the treatment efficacy. The results of breast cancer oxygen state study under chemotherapy action obtained by diffuse optical spectroscopy (DOS) in combination with Doppler ultrasonic imaging are presented. Complex use of optical and ultrasound methods gives complementary information about the size of the tumor node, peculiarities of its vascular bed, rate of its blood flow as well as oxygenation, and provide a picture of the tumor response to treatment. Comparison with tumor pathologic response allowed to identify differences in the changes of these parameters depending on the degree of pathological tumor response to chemotherapy. It was demonstrated that fourth and fifth degrees of therapeutic pathomorphism may be predicted by the increase of oxygen saturation level after the first cycle of chemotherapy. If the reduction or absence of the oxygen saturation dynamics is observed, first or second degree of pathological tumor response can be expected. Additional ultrasound investigation of the tumors may be useful for observation of the dynamics of tumor blood flow thereby for understanding the reasons of induced chemotherapy oxygenation changes. The proposed approach based on DOS and ultrasonography may be applied for monitoring of breast tumors under therapy and prediction of their sensitivity.

Mitochondria-Targeted Antioxidant SkQ1 Improves Dermal Wound Healing in Genetically Diabetic Mice.

Oxidative stress is widely recognized as an important factor in the delayed wound healing in diabetes. However, the role of mitochondrial reactive oxygen species in this process is unknown. It was assumed that mitochondrial reactive oxygen species are involved in many wound-healing processes in both diabetic humans and animals. We have applied the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to explore the role of mitochondrial reactive oxygen species in the wound healing of genetically diabetic mice. Healing of full-thickness excisional dermal wounds in diabetic C57BL/KsJ-db-/db- mice was significantly enhanced after long-term (12 weeks) administration of SkQ1. SkQ1 accelerated wound closure and stimulated epithelization, granulation tissue formation, and vascularization. On the 7th day after wounding, SkQ1 treatment increased the number of α-smooth muscle actin-positive cells (myofibroblasts), reduced the number of neutrophils, and increased macrophage infiltration. SkQ1 lowered lipid peroxidation level but did not change the level of the circulatory IL-6 and TNF. SkQ1 pretreatment also stimulated cell migration in a scratch-wound assay in vitro under hyperglycemic condition. Thus, a mitochondria-targeted antioxidant normalized both inflammatory and regenerative phases of wound healing in diabetic mice. Our results pointed to nearly all the major steps of wound healing as the target of excessive mitochondrial reactive oxygen species production in type II diabetes.

Mitochondria-targeted antioxidant SkQ1 improves impaired dermal wound healing in old mice.

The process of skin wound healing is delayed or impaired in aging animals. To investigate the possible role of mitochondrial reactive oxygen species (mtROS) in cutaneous wound healing of aged mice, we have applied the mitochondria-targeted antioxidant SkQ1. The SkQ1 treatment resulted in accelerated resolution of the inflammatory phase, formation of granulation tissue, vascularization and epithelization of the wounds. The wounds of SkQ1-treated mice contained increased amount of myofibroblasts which produce extracellular matrix proteins and growth factors mediating granulation tissue formation. This effect resembled SkQ1-induced differentiation of fibroblasts to myofibroblast, observed earlierin vitro. The Transforming Growth Factor beta (TGFb) produced by SkQ1-treated fibroblasts was found to stimulated motility of endothelial cells in vitro, an effect which may underlie pro-angiogenic action of SkQ1 in the wounds. In vitro experiments showed that SkQ1 prevented decomposition of VE-cadherin containing contacts and following increase in permeability of endothelial cells monolayer, induced by pro-inflammatory cytokine TNF. Prevention of excessive reaction of endothelium to the pro-inflammatory cytokine(s) might account for anti-inflammatory effect of SkQ1. Our findings point to an important role of mtROS in pathogenesis of age-related chronic wounds.

A novel secreted metzincin metalloproteinase from Bacillus intermedius.

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.

Smad expression in human atherosclerotic lesions: evidence for impaired TGF-beta/Smad signaling in smooth muscle cells of fibrofatty lesions.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of human atherosclerosis but its actions during lesion progression are poorly understood. Smad2, Smad3, and Smad4 proteins are signaling molecules by which TGF-beta modulates gene transcription. Our objective was to define the actions of TGF-beta during lesion progression in humans by examining the expression of Smads in relation to TGF-beta-mediated responses. METHODS AND RESULTS: Immunohistochemistry and reverse-transcription polymerase chain reaction demonstrated Smad2, Smad3, and Smad4 expression in macrophages of fibrofatty lesions and their upregulation after differentiation of monocytes to macrophages. The major Smad splice variants expressed by the macrophages were those that are transcriptionally most active. Macrophages also expressed cyclin inhibitors whose expression is induced via Smad proteins. The cytoplasmic location of p21(Waf1) suggests it may protect macrophages from apoptosis. Smooth muscle cells (SMCs) within the fibrofatty lesions did not express the Smad proteins or the cyclin inhibitors. SMCs of fibrous plaques expressed all 3 Smad proteins. CONCLUSIONS: In human atherosclerotic lesions, the actions of TGF-beta appear restricted to SMCs in fibrous plaques and macrophages in fatty streaks/fibrofatty lesions. The lack of key TGF-beta signaling components in SMCs of fibrofatty lesions indicates impaired ability of these cells to initiate TGF-beta-mediated Smad-dependent transcriptional responses.

Cytochrome b558-dependent NAD(P)H oxidase-phox units in smooth muscle and macrophages of atherosclerotic lesions.

OBJECTIVE: Despite studies implicating superoxide anion-producing oxidases in atherosclerosis, their characteristics, expression, and regulation in cells of lesions are poorly understood. We examined the following: (1) whether cytochrome b558-dependent NAD(P)H oxidase-phox peptides are expressed by intimal smooth muscle cells (iSMCs) and macrophages of human aortic atherosclerotic lesions and their regulation and (2) whether cytochrome b558-dependent NAD(P)H oxidase represents a major NAD(P)H oxidase in iSMCs. METHODS AND RESULTS: Using a combination of immunochemical and reverse transcription-polymerase chain reaction procedures, we demonstrate that p22(phox) and gp91(phox) (cytochrome b558) expression in normal intima was restricted to a quarter of the iSMCs. In fatty streaks, a similar fraction of iSMCs expressed cytochrome b558, whereas macrophages also expressed low levels of p47(phox) and p67(phox). In fibrofatty lesions, the majority of iSMCs expressed the cytochrome b558 subunits; p67(phox) was also detected. Macrophages and macrophage-derived foam cells expressed the 4 phox subunits that constitute superoxide-producing cytochrome b558-dependent NAD(P)H oxidase. These were upregulated by transforming growth factor-beta1 and interferon-gamma. Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91(phox). CONCLUSIONS: In human aortic fibrofatty lesions, a cytochrome b558-dependent NAD(P)H oxidase appears to be a major iSMC and macrophage oxidase whose expression is upregulated by cytokines.

Distribution, phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection.

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.