Real-time calculation of a limiting form of the Renyi entropy applied to detection of subtle changes in scattering architecture.

Previously a new method for ultrasound signal characterization using entropy H(f) was reported, and it was demonstrated that in certain settings, further improvements in signal characterization could be obtained by generalizing to Renyi entropy-based signal characterization I(f)(r) with values of r near 2 (specifically r=1.99) [M. S. Hughes et al., J. Acoust. Soc. Am. 125, 3141-3145 (2009)]. It was speculated that further improvements in sensitivity might be realized at the limit r-->2. At that time, such investigation was not feasible due to excessive computational time required to calculate I(f)(r) near this limit. In this paper, an asymptotic expression for the limiting behavior of I(f)(r) as r-->2 is derived and used to present results analogous to those obtained with I(f)(1.99). Moreover, the limiting form I(f,infinity) is computable directly from the experimentally measured waveform f(t) by an algorithm that is suitable for real-time calculation and implementation.

Application of Renyi entropy for ultrasonic molecular imaging.

Previous work has demonstrated that a signal receiver based on a limiting form of the Shannon entropy is, in certain settings, more sensitive to subtle changes in scattering architecture than conventional energy-based signal receivers [M. S. Hughes et al., J. Acoust. Soc. Am. 121, 3542-3557 (2007)]. In this paper new results are presented demonstrating further improvements in sensitivity using a signal receiver based on the Renyi entropy.

Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging.

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha.

Hypoxia-inducible factor-1alpha (HIF-1alpha) transactivates genes required for energy metabolism and tissue perfusion and is necessary for embryonic development and tumor explant growth. HIF-1alpha is overexpressed during carcinogenesis, myocardial infarction, and wound healing; however, the biological consequences of HIF-1alpha overexpression are unknown. Here, transgenic mice expressing constitutively active HIF-1alpha in epidermis displayed a 66% increase in dermal capillaries, a 13-fold elevation of total vascular endothelial growth factor (VEGF) expression, and a six- to ninefold induction of each VEGF isoform. Despite marked induction of hypervascularity, HIF-1alpha did not induce edema, inflammation, or vascular leakage, phenotypes developing in transgenic mice overexpressing VEGF cDNA in skin. Remarkably, blood vessel leakage resistance induced by HIF-1alpha overexpression was not caused by up-regulation of angiopoietin-1 or angiopoietin-2. Hypervascularity induced by HIF-1alpha could improve therapy of tissue ischemia.

Coordinate up-regulation of hypoxia inducible factor (HIF)-1alpha and HIF-1 target genes during multi-stage epidermal carcinogenesis and wound healing.

Both carcinogenesis and wound healing proceed through stages of proliferation and tissue remodeling. Here, using either a model of multistage epidermal carcinogenesis in K14-HPV16 transgenic mice or creation of full-thickness back wounds in nontransgenic mice, we determined patterns of expression of hypoxia inducible factor (HIF)-1alpha, and three targets of the heterodimeric transcription factor HIF-1, glucose transporter (GLUT)-1, phosphoglycerate kinase (PGK)-1, and vascular endothelial growth factor (VEGF) in skin. Neither HIF-1alpha, GLUT-1, PGK-1, nor VEGF mRNA was detectable in unwounded nontransgenic skin. In epidermal carcinogenesis, HIF-1alpha, GLUT-1, PGK-1, and VEGF mRNAs were just detectable in early-stage hyperplasia, markedly increased in high-grade epidermal chest dysplasias, and further increased in invasive squamous carcinomas. In neoplastic skin, HIF-1alpha, GLUT-1, and PGK-1 mRNAs localized in the basal and immediate suprabasal epidermal layers, whereas VEGF mRNA was predominantly expressed in the more superior spinous and granular epidermal layers. Immediately after wounding, HIF-1alpha, GLUT-1, and PGK-1 mRNAs were detectable in basal keratinocytes at the wound edge. Expression of all three genes increased to maximum levels in reepithelializing basal keratinocytes and then diminished to near undetectable levels after wound epithelialization. Although VEGF mRNA similarly increased and decreased during wound healing, its expression pattern was more punctate; the most intense hybridization signals were detected in the upper spinous and granular layers of reepithelializing keratinocytes and in dermal cells morphologically similar to macrophages. These data suggest stage-specific and spatio-temporal control of HIF-1alpha and HIF-1 target gene expression in both multistage epithelial carcinogenesis and wound healing.

Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth.

Deficiencies in oxygenation are widespread in solid tumors. The transcription factor hypoxia-inducible factor (HIF)-1alpha is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors important for solid tumor expansion, including the angiogenic factor vascular endothelial growth factor (VEGF). We have isolated two cell lines nullizygous for HIF-1alpha, one from embryos genetically null for HIF-1alpha, and the other from embryos carrying loxP-flanked alleles of the gene, which allows for cre-mediated excision. The loss of HIF-1alpha negatively affects tumor growth in these two sets of H-ras-transformed cell lines, and this negative effect is not due to deficient vascularization. Despite differences in VEGF expression, vascular density is similar in wild-type and HIF-1alpha-null tumors. The evidence from these experiments indicates that hypoxic response via HIF-1alpha is an important positive factor in solid tumor growth and that HIF-1alpha affects tumor expansion in ways unrelated to its regulation of VEGF expression.

Altered basic fibroblast growth factor expression in common epidermal neoplasms: examination with in situ hybridization and immunohistochemistry.

BACKGROUND: The fibroblast growth factor family consists of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and Kaposi fibroblast growth factor (kFGF). The distribution of these growth factors in skin disease has not been determined. OBJECTIVE: The purpose of this study was to determine the distribution of these growth factors in keratinocytic lesions and normal skin. METHODS: Skin sections from common disorders of keratinocytes were examined by in situ hybridization with specific probes for aFGF, bFGF, and kFGF, and immunohistochemistry. RESULTS: Of the growth factors studied, only bFGF was present in skin. bFGF messenger RNA was highly expressed in both normal epidermis and benign and malignant epithelial neoplasms. In normal skin, bFGF was expressed predominantly in a suprabasal fashion, whereas in epithelial neoplasms, homogeneous high-level expression of bFGF was observed. CONCLUSION: bFGF is the primary member of the fibroblast growth factor expressed in the skin. The source of synthesis of bFGF is keratinocytes. Immunoreactivity for bFGF appears to be primarily confined to upper layers of the epidermis in normal skin, but is expressed at all layers of the epidermis in both benign and malignant neoplastic conditions. Genetic changes that promote epithelial tumors may also promote translation of bFGF messenger RNA into protein. Specific inhibition of bFGF activity may have application in the treatment of common skin diseases.

Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis.

Regions where one type of epithelium replaces another (metaplasia) have a predilection for cancer formation. Environmental factors are closely linked to metaplastic carcinogenesis. In particular, cervical cancers associated with human papillomavirus (HPV) infection develop primarily at the transformation zone, a region where metaplastic squamous cells are detected in otherwise columnar epithelial-lined endocervical glands. Previously, we reported estrogen-induced multistage vaginal and cervical carcinogenesis in transgenic mice expressing HPV16 oncogenes in basal squamous epithelial cells. In the present study to investigate the threshold neoplastic response to exogenous estrogen, we treated groups of transgenic mice with lower hormone doses. A 5-fold reduction in estrogen dose induced squamous carcinogenesis solely at the cervical transformation zone compared with other reproductive tract sites. Further study delineated stages of transformation zone carcinogenesis, including formation of hyperplastic lower uterine glands and emergence of multiple foci of squamous metaplasia from individual stem-like glandular reserve cells, followed by neoplastic progression of metaplasia to dysplasia and squamous cancer. We propose that a combination of low-dose estrogen and low-level HPV oncogene expression biases transformation zone glandular reserve cells toward squamous rather than columnar epithelial fate decisions. Synergistic activation of proliferation by viral oncoprotein cell cycle dysregulation and estrogen receptor signaling, together with altered paracrine stromal-epithelial interactions, may conspire to support and promote neoplastic progression and cancer formation.

Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E.

The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components, Skp1, Cul-1 (Cdc53 in yeast) and Rbx1, and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation. To study the role of Cul-1 in mammalian development and cell-cycle regulation, we generated mice deficient for Cul1 and analysed null embryos and heterozygous cell lines. We show that Cul1 is required for early mouse development and that Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis.

Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice.

Mice that express transgenes for human papillomavirus type 16 under a keratin 14 promoter (K14-HPV16 mice) develop cervical cancer when they are given 17beta-estradiol chronically. We asked whether the antiestrogenic phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, administered at physiological doses, would prevent the cervical-vaginal cancer that is promoted in these mice by high doses of estrogen. We compared mice that were fed a control diet with those that were fed a diet supplemented with 2000 ppm I3C. In the group fed the control diet, at a dose of estradiol of 0.125 mg per 60-day release, 19 of 25 transgenic mice developed cervical-vaginal cancer within 6 months, and the remainder had dysplasia. Only 2 mice of 24 in the group fed the I3C supplemented diet developed cancer, and the remainder had dysplasia or hyperplasia. I3C reduced dysplasia in the nontransgenic mice. Similar results were obtained at a higher dose of estradiol (0.250 mg per 60-day release), and I3C helped to prevent morbidity associated with retention of fluid in the bladder that frequently occurred with the higher estradiol dose. Additionally, I3C appeared to reduce skin cancer in transgenic mice. These data indicate that I3C is a useful preventive for cervical-vaginal cancer and, possibly, other cancers with a papillomavirus component.

Difluoromethylornithine chemoprevention of epidermal carcinogenesis in K14-HPV16 transgenic mice.

To be informative for chemoprevention, animal models must both closely emulate human disease and possess surrogate endpoint biomarkers that facilitate rapid drug screening. This study elucidated site-specific histopathological and biochemical surrogate endpoint biomarkers of spontaneous epidermal carcinogenesis in K14-HPV16 transgenic mice and demonstrated that the incidence and severity of these markers were decreased by the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). The cumulative incidence of visible epidermal cancers in 127 untreated transgenic mice was 42% by 52 weeks of age, most frequently affecting the chest as flat lesions in association with chronic ulcers, or in the ear as protuberant masses. Microscopic malignancies were detected in 39% of 32-week-old transgenic mice and were found to emerge from precursor lesions that were of two distinct types: dysplastic sessile ear papillomas and hyperproliferative follicular/interfollicular chest dysplasias. ODC activity and tissue polyamine contents were differentially elevated in ear and chest skin during carcinogenesis, such that there was a marked elevation of both parameters of polyamine metabolism as early as 4 weeks of age in the ear, whereas in the chest, polyamine metabolism was increased significantly only in the late stages of neoplastic progression and in epidermal cancers. Administration of 1.0% DFMO in the drinking water from 4 to 32 weeks of age prevented both visible and microscopic malignancies and significantly decreased the incidence of chest and ear precursor lesions. ODC activity and tissue putrescine content were markedly diminished by DFMO chemoprevention in ear skin, whereas there was a more modest decline of these parameters in chest skin. DFMO treatment of transgenic mice from 28 to 32 weeks of age was associated with an absence of ear cancer and a marked regression of dysplastic papillomas. In contrast, the results in chest skin were complex in that the severity of chest precursors diminished, but their incidence was unchanged, and microscopic cancers were still detectable within these lesions. Collectively, this study highlights the utility of multistage epidermal carcinogenesis in K14-HPV16 transgenic mice both for the study of the biology of, and as a screening tool for, novel drugs and chemopreventive regimens.

Expression of interferon-beta is associated with growth arrest of murine and human epidermal cells.

The cytokine interferon-beta is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-beta by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-beta protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-beta whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-beta protein. Neutralizing interferon-beta in the culture medium inhibited differentiation, but the addition of exogenous interferon-beta did not stimulate differentiation. These data indicate that interferon-beta is produced by growth-arrested, terminally differentiated keratinocytes.

Murine mentors: transgenic and knockout models of surgical disease.

OBJECTIVE: Transgenic and knockout technologies have emerged from the "molecular biology revolution" as unprecedented techniques for manipulating gene function in intact mice. The goals of this review are to outline the techniques of creating transgenic and knockout mice, and to demonstrate their use in elucidation of the molecular mechanisms underlying common surgical diseases. SUMMARY BACKGROUND DATA: Gain of gene function is created by transgenic technology, whereas gene function is ablated using gene knockouts. Each technique has distinctive applications and drawbacks. A unique feature of genetically manipulated mice is that combinatorial genetic experiments can be executed that precisely define the functional contribution of a gene to disease progression. Transgenic and knockout mouse models of wound healing, cardiovascular disease, transplant immunology, gut motility and inflammatory bowel disease, and oncology are beginning to illuminate the precise molecular regulation of these diseases. Transgenic technology has also been extended to larger mammals such as pigs, with the goal of using genetic manipulation of the xenogenic immune response to increase the availability of transplant organs. Continual refinements in gene manipulation technology in mice offer the opportunity to turn genes on or off at precise time intervals and in particular tissues, according to the needs of the investigator. Ultimately, investigation of disease development and progression in genetically manipulated mammals may delineate new molecular targets for drug discovery and provide novel platforms for drug efficacy screens. CONCLUSIONS: Emulation of human disease and therapy using genetically manipulated mammals fulfills a promise of molecular medicine: fusion of molecular biochemistry with "classical" biology and physiology. Surgeons have unique skills spanning both worlds that can facilitate their success in this expanding arena.

Modulation of the human homeobox genes PRX-2 and HOXB13 in scarless fetal wounds.

Scarless healing of cutaneous wounds occurs in humans during the first two trimesters of development, but by birth all wounds are repaired with scar formation. To search for transcriptional regulatory genes that might mediate fetal tissue regeneration, we surveyed homeobox gene expression in proliferating fetal fibroblasts and in wounded and unwounded skin. Two novel human homeobox genes, PRX-2 and HOXB13, were identified that were differentially expressed during fetal versus adult wound healing. Both genes were predominantly expressed in proliferating fetal fibroblasts and developing dermis, and PRX-2 was downregulated in adult skin. In a model of scarless fetal skin regeneration, PRX-2 expression was strongly increased compared with unwounded skin and the signal was localized to the wounded dermis, the site of scarless repair. Conversely, in adult skin weak epidermal PRX-2 expression was observed, mRNA levels were not increased by wounding, and no dermal expression was detected. HOXB13 expression was decreased in wounded fetal tissue relative to unwounded fetal controls or wounded adult skin. Thus both HOXB13 and PRX-2 are expressed in patterns consistent with roles in fetal skin development and cutaneous regeneration.

Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice.

Infection of the human cervix with certain papillomavirus subtypes is associated with the development of neoplastic squamous lesions that can progress to overt cervical malignancies. Recently, multistage squamous carcinogenesis has been achieved in K14-HPV16 transgenic mice, wherein expression of the human papillomavirus (HPV) type 16 early genes is targeted to basal squamous epithelial cells by regulatory elements of the human keratin-14 (K14) promoter. Immunostaining of the endothelial marker vWf revealed a parallel upregulation of angiogenesis during the early neoplastic stages in both human cervix and the epidermis of K14-HPV16 transgenic mice. Moreover, high-grade premalignant lesions and cancers in humans and transgenic mice were characterized by an additional increment in the number of new capillaries and close apposition of the microvasculature to the overlying neoplastic epithelium. Expression of the potent angiogenic factor VEGF was progressively up-regulated during carcinogenesis in both species, correlating with the increased density and altered distribution of the microvasculature. Thus, angiogenesis occurs during the premalignant stages of squamous carcinogenesis in both human cervical disease and a relevant transgenic model and may be controlled by similar molecular mechanisms in both species. These results validate the use of the transgenic model to elucidate the role of angiogenesis during HPV-associated neoplastic progression.

Human acellular dermal matrix as a novel model of malignant epithelial cell invasion.

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.

Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.

Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.

Upregulation of fibroblast growth factors and their receptors during multi-stage epidermal carcinogenesis in K14-HPV16 transgenic mice.

Upregulation of acidic and basic fibroblast growth factors (FGF-1 and -2), and their cognate receptors FGFR-1 and -2, has been demonstrated in a variety of epithelial malignancies. However, the patterns of FGF/FGFR expression at specific stages of epithelial carcinogenesis have not been extensively characterized. In this report, the levels of FGF-1, FGF-2, FGF-7 mRNA and their receptors FGFR-1 and FGFR-2, were investigated during epidermal carcinogenesis in transgenic mice expressing the early region of the 'high risk' papillomavirus type 16 (HPV16) under control of the human keratin-14 enhancer/promoter (K14-HPV16 transgenic mice). FGF-1 was first upregulated in dysplasias, while FGF-2 was constitutively expressed in non-transgenic, neoplastic, and malignant keratinocytes throughout carcinogenesis. Expression of FGF-7 was undetectable in non-transgenic epidermis, and remained at threshold levels at all stages of progression. In well differentiated squamous cancers, FGFR-1 was upregulated and co-localized with angiogenic capillaries in the dermis underlying dysplastic lesions and within papillary fronds of invasive cancers. In contrast, FGFR-1 was upregulated specifically within the malignant squamous cells of moderate-poorly differentiated squamous cancers. The expression of FGFR-2 was essentially constitutive in both non-transgenic and neoplastic epidermis. Collectively the data suggest that the FGF/FGFR signaling pathways may potentially contribute to several facets of multi-stage epithelial carcinogenesis, including auto- or paracrine growth stimulation, upregulation of angiogenesis, and stromal remodeling.