Modeling the communication-satisfaction relationship in hospital patients.

OBJECTIVES: Although it has long been known that communication with medical professionals presents a strong relationship with patient satisfaction, research on this topic has been hindered by conceptual and methodological issues (e.g. single-item measures, inclusion of idiosyncratic patient characteristics, etc.). Using a more comprehensive and integrated approach, this study had two objectives: to document the multidimensional structure of the Picker Patient Experience-15, and to test a patient communication/satisfaction model that organizes its dimensions in a conceptually logical array of relationships. First, the factorial structure of the Picker Patient Experience-15 was hypothesized to comprise five dimensions: communication with patient, with family, addressing fears/concerns, preparation for discharge, and patient satisfaction. Second, the hypothesized model included positive relationships between all four communications dimensions, on the one hand, and patient satisfaction, on the other. Within communication dimensions, communication with patient was hypothesized to be the incipient factor for other dimensions, and thus to be positively associated with the other three forms of communication. METHODS: This research is based on a single time point design, which relied on administrative and questionnaire data. The study was conducted at a large University Hospital in Switzerland. The sample included 54,686 patients who received inpatient treatment, excluding those who were cared for in the intensive and intermediate care units. Patients filled out, over a 5-year period, the Picker Patient Experience questionnaire (PPE-15) after discharge (overall response rate of 41%). RESULTS: The proposed five-factor structure of the Picker Patient Experience-15 was successfully supported by the results of a confirmatory factor analysis. Moreover, the hypothesized network of associations between communication and satisfaction latent constructs was substantiated using structural equation modeling. With the exception of the association between preparation for discharge and patient satisfaction, the hypothesized model was fully corroborated. CONCLUSION: A more in-depth understanding of patient satisfaction can be achieved when it is studied as a multifaceted phenomenon.

Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First-in-Man Study.

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic- and vascular-progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking-plate osteosynthesis, with cell-free grafts as control. After 8 weeks, only SVF-treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low-energy proximal humeral fractures in 8 patients (64-84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture-microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra-operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956-2966.

Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle.

Therapeutic angiogenesis by growth factor delivery is an attractive treatment strategy for ischemic diseases, yet clinical efficacy has been elusive. The angiogenic master regulator VEGF-A can induce aberrant angiogenesis if expressed above a threshold level. Since VEGF remains localized in the matrix around expressing cells, homogeneous dose distribution in target tissues is required, which is challenging. We found that co-expression of the pericyte-recruiting factor PDGF-BB at a fixed ratio with VEGF from a single bicistronic vector ensured normal angiogenesis despite heterogeneous high VEGF levels. Taking advantage of a highly controlled gene delivery platform, based on monoclonal populations of transduced myoblasts, in which every cell stably produces the same amount of each factor, here we rigorously investigated a) the dose-dependent effects, and b) the long-term safety and stability of VEGF and PDGF-BB co-expression in skeletal muscle. PDGF-BB co-expression did not affect the normal angiogenesis by low and medium VEGF doses, but specifically prevented vascular tumors by high VEGF, yielding instead normal and mature capillary networks, accompanied by robust arteriole formation. Induced angiogenesis persisted unchanged up to 4 months, while no tumors appeared. Therefore, PDGF-BB co-expression is an attractive strategy to improve safety and efficacy of therapeutic angiogenesis by VEGF gene delivery.

CD40 ligand-expressing recombinant vaccinia virus promotes the generation of CD8(+) central memory T cells.

Central memory CD8(+) T cells (TCM ) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8(+) TCM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8(+) TCM -cell generation is challenging, and usually requires CD4(+) CD40L(+) T-cell "help" during the priming of naïve CD8(+) T cells. We have generated a replication incompetent CD40 ligand-expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8(+) T cells into TCM specific for viral and tumor-associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild-type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4(+) T cells, a single "in vitro" stimulation of naïve CD8(+) T cells by rVV40L-infected nonprofessional CD14(+) antigen presenting cells promotes the rapid generation of viral or tumor associated antigen-specific CD8(+) T cells displaying TCM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8(+) mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.

Absence of myeloperoxidase and CD8 positive cells in colorectal cancer infiltrates identifies patients with severe prognosis.

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPOhigh/CD8low or MPOlow/CD8high infiltrates. Most importantly, we identified a subgroup of CRC with MPOlow/CD8low tumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-ß1 paracrine axis.

VEGF is widely investigated for therapeutic angiogenesis, but while short-term delivery is desirable for safety, it is insufficient for new vessel persistence, jeopardizing efficacy. Here, we investigated whether and how VEGF dose regulates nascent vessel stabilization, to identify novel therapeutic targets. Monoclonal populations of transduced myoblasts were used to homogeneously express specific VEGF doses in SCID mouse muscles. VEGF was abrogated after 10 and 17 days by Aflibercept treatment. Vascular stabilization was fastest with low VEGF, but delayed or prevented by higher doses, without affecting pericyte coverage. Rather, VEGF dose-dependently inhibited endothelial Semaphorin3A expression, thereby impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-ß1 production and endothelial SMAD2/3 activation. TGF-ß1 further initiated a feedback loop stimulating endothelial Semaphorin3A expression, thereby amplifying the stabilizing signals. Blocking experiments showed that NEM recruitment required endogenous Semaphorin3A and that TGF-ß1 was necessary to start the Semaphorin3A/NEM axis. Conversely, Semaphorin3A treatment promoted NEM recruitment and vessel stabilization despite high VEGF doses or transient adenoviral delivery. Therefore, VEGF inhibits the endothelial Semaphorin3A/NEM/TGF-ß1 paracrine axis and Semaphorin3A treatment accelerates stabilization of VEGF-induced angiogenesis.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed "in vivo".

Anticancer compound screening on 2D cell cultures poorly predicts "in vivo" performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only "nucleolar stress" in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed "in vivo" and to test anticancer compounds.

Designing questionnaires: healthcare survey to compare two different response scales.

BACKGROUND: A widely discussed design issue in patient satisfaction questionnaires is the optimal length and labelling of the answering scale. The aim of the present study was to compare intra-individually the answers on two response scales to five general questions evaluating patients' perception of hospital care. METHODS: Between November 2011 and January 2012, all in-hospital patients at a Swiss University Hospital received a patient satisfaction questionnaire on an adjectival scale with three to four labelled categories (LS) and five redundant questions displayed on an 11-point end-anchored numeric scale (NS). The scales were compared concerning ceiling effect, internal consistency (Cronbach's alpha), individual item answers (Spearman's rank correlation), and concerning overall satisfaction by calculating an overall percentage score (sum of all answers related to the maximum possible sum). RESULTS: The response rate was 41% (2957/7158), of which 2400 (81%) completely filled out all questions. Baseline characteristics of the responders and non-responders were similar. Floor and ceiling effect were high on both response scales, but more pronounced on the LS than on the NS. Cronbach's alpha was higher on the NS than on the LS. There was a strong individual item correlation between both answering scales in questions regarding the intent to return, quality of treatment and the judgement whether the patient was treated with respect and dignity, but a lower correlation concerning satisfactory information transfer by physicians or nurses, where only three categories were available in the LS. The overall percentage score showed a comparable distribution, but with a wider spread of lower satisfaction in the NS. CONCLUSIONS: Since the longer scale did not substantially reduce the ceiling effect, the type of questions rather than the type of answering scale could be addressed with a focus on specific questions about concrete situations instead of general questions. Moreover, the low correlation in questions about information provision suggests that only three possible response choices are insufficient. Further investigations are needed to find a more sensitive scale discriminating high-end ratings. Otherwise, a longitudinal within-hospital or a cross-sectional between-hospital comparison of patient care is questionable.

Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial.

BACKGROUND: Autologous native cartilage from the nasal septum, ear, or rib is the standard material for surgical reconstruction of the nasal alar lobule after two-layer excision of non-melanoma skin cancer. We assessed whether engineered autologous cartilage grafts allow safe and functional alar lobule restoration. METHODS: In a first-in-human trial, we recruited five patients at the University Hospital Basel (Basel, Switzerland). To be eligible, patients had to be aged at least 18 years and have a two-layer defect (≥50% size of alar subunit) after excision of non-melanoma skin cancer on the alar lobule. Chondrocytes (isolated from a 6 mm cartilage biopsy sample from the nasal septum harvested under local anaesthesia during collection of tumour biopsy sample) were expanded, seeded, and cultured with autologous serum onto collagen type I and type III membranes in the course of 4 weeks. The resulting engineered cartilage grafts (25 mm × 25 mm × 2 mm) were shaped intra-operatively and implanted after tumour excision under paramedian forehead or nasolabial flaps, as in standard reconstruction with native cartilage. During flap refinement after 6 months, we took biopsy samples of repair tissues and histologically analysed them. The primary outcomes were safety and feasibility of the procedure, assessed 12 months after reconstruction. At least 1 year after implantation, when reconstruction is typically stabilised, we assessed patient satisfaction and functional outcomes (alar cutaneous sensibility, structural stability, and respiratory flow rate). FINDINGS: Between Dec 13, 2010, and Feb 6, 2012, we enrolled two women and three men aged 76-88 years. All engineered grafts contained a mixed hyaline and fibrous cartilage matrix. 6 months after implantation, reconstructed tissues displayed fibromuscular fatty structures typical of the alar lobule. After 1 year, all patients were satisfied with the aesthetic and functional outcomes and no adverse events had been recorded. Cutaneous sensibility and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function. INTERPRETATION: Autologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions. FUNDING: Foundation of the Department of Surgery, University Hospital Basel; and Krebsliga beider Basel.

Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-ß.

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-ß newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

The relationship between hospital patients' ratings of quality of care and communication.

OBJECTIVE: To assess the relationship between hospital patients' quality of care ratings and their experiences with health-related information exchanges and communication during hospitalization. DESIGN: Cross-sectional multivariate dimensional analysis of data from a quality of care experience questionnaire of hospital patients comparing scores across three levels of reported satisfaction. SETTING AND PARTICIPANTS: Five thousand nine hundred and fifty-two patients from a Swiss University Hospital responded to the questionnaire at discharge during 2010. MAIN OUTCOME MEASURES: Survey questions measuring patients' evaluation of quality of care, patient loyalty and overall satisfaction. RESULTS: Different levels of reported satisfaction are associated with differing experiences of health-related information and communication during a hospital stay. CONCLUSIONS: Patients who report lower satisfaction appear to attribute to the hospital staff enduring negative dispositions from behaviours that may be due to specific situational contexts. Negative experiences appear to influence scores on most other communication and information domains. Patients who report higher satisfaction, in contrast, appear to differentiate negative experiences and positive experiences and they appear to relativize and compartmentalize negative experiences associated with their hospital stay.

High frequency of CD8 positive lymphocyte infiltration correlates with lack of lymph node involvement in early rectal cancer.

AIMS: A trend towards local excision of early rectal cancers has prompted us to investigate if immunoprofiling might help in predicting lymph node involvement in this subgroup. METHODS: A tissue microarray of 126 biopsies of early rectal cancer (T1 and T2) was stained for several immunomarkers of the innate and the adaptive immune response. Patients' survival and nodal status were analyzed and correlated with infiltration of the different immune cells. RESULTS: Of all tested markers, only CD8 (P = 0.005) and TIA-1 (P = 0.05) were significantly more frequently detectable in early rectal cancer biopsies of node negative as compared to node positive patients. Although these two immunomarkers did not display prognostic effect "per se," CD8+ and, marginally, TIA-1 T cell infiltration could predict nodal involvement in univariate logistic regression analysis (OR 0.994; 95% CI 0.992-0.996; P = 0.009 and OR 0.988; 95% CI 0.984-0.994; P = 0.05, resp.). An algorithm significantly predicting the nodal status in early rectal cancer based on CD8 together with vascular invasion and tumor border configuration could be calculated (P < 0.00001). CONCLUSION: Our data indicate that in early rectal cancers absence of CD8+ T-cell infiltration helps in predicting patients' nodal involvement.

Characterization and clinical relevance of ALDHbright populations in prostate cancer.

PURPOSE: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS: ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor.

BACKGROUND: Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). METHODS: A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. RESULTS: MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO-. CONCLUSIONS: High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.

Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors.

Rapid vascularisation of tissue-engineered osteogenic grafts is a major obstacle in the development of regenerative medicine approaches for bone repair. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth. We investigated a cell-based gene therapy approach to generate osteogenic grafts with an increased vascularization potential in an ectopic nude rat model in vivo, by genetically modifying human bone marrow-derived stromal/stem cells (BMSC) to express rat VEGF. BMSC were loaded onto silicate-substituted apatite granules, which are a clinically established osteo-conductive material. Eight weeks after implantation, the vascular density of constructs seeded with VEGF-BMSC was 3-fold greater than with control cells, consisting of physiologically structured vascular networks with both conductance vessels and capillaries. However, VEGF specifically caused a global reduction in bone quantity, which consisted of thin trabeculae of immature matrix. VEGF did not impair BMSC engraftment in vivo, but strongly increased the recruitment of TRAP- and Cathepsin K-positive osteoclasts. These data suggest that VEGF over-expression is effective to improve the vascularization of osteogenic grafts, but also has the potential to disrupt bone homoeostasis towards excessive degradation, posing a challenge to its clinical application in bone tissue engineering.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND: Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS: A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS: Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION: PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Induction of aberrant vascular growth, but not of normal angiogenesis, by cell-based expression of different doses of human and mouse VEGF is species-dependent.

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery is an attractive approach to treat ischemia. VEGF remains localized around each producing cell in vivo, and overexpression of mouse VEGF(164) (mVEGF(164)) induces normal or aberrant angiogenesis, depending strictly on its dose in the microenvironment in vivo. However, the dose-dependent effects of the clinically relevant factor, human VEGF(165) (hVEGF(165)), are unknown. Here we exploited a highly controlled gene delivery platform, based on clonal populations of transduced myoblasts overexpressing specific VEGF levels, to rigorously compare the in vivo dose-dependent effects of hVEGF(165) and mVEGF(164) in skeletal muscle of severe combined immune deficient (SCID) mice. While low levels of both factors efficiently induced similar amounts of normal angiogenesis, only high levels of mVEGF(164) caused widespread angioma-like structures, whereas equivalent or even higher levels of hVEGF(165) induced exclusively normal and mature capillaries. Expression levels were confirmed both in vitro and in vivo by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). However, in vitro experiments showed that hVEGF(165) was significantly more effective in activating VEGF receptor signaling in human endothelial cells than mVEGF(164), while the opposite was true in murine endothelial cells. In conclusion, we found that, even though hVEGF is similarly efficient to the syngenic mVEGF in inducing angiogenesis at lower doses in a widely adopted and convenient mouse preclinical model, species-dependent differences in the relative activation of the respective receptors may specifically mask the toxic effects of high doses of the human factor.

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting.

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells.

BACKGROUND: Cancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA). METHODS: Fifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples. RESULTS: Nanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4α isoform. CONCLUSION: Klf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.

CD133+, CD166+CD44+, and CD24+CD44+ phenotypes fail to reliably identify cell populations with cancer stem cell functional features in established human colorectal cancer cell lines.

Increasing evidence that cancers originate from small populations of so-called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC-targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high-throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC-specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell-like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase-1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC-specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery.