Role of Gut Microbiome in Neoadjuvant Chemotherapy Response in Urothelial Carcinoma: A Multi-institutional Prospective Cohort Evaluation.

Neoadjuvant chemotherapy (NAC) is linked with clinical advantages in urothelial carcinoma for patients with muscle-invasive bladder cancer (MIBC). Despite comprehensive research into the influence of tumor mutation expression profiles and clinicopathologic factors on chemotherapy response, the role of the gut microbiome (GM) in bladder cancer chemotherapy response remains poorly understood. This study examines the variance in the GM of patients with bladder cancer compared with healthy adults, and investigates GM compositional differences between patients who respond to chemotherapy versus those who exhibit residual disease.Our study reveals distinct clustering, effectively separating the bladder cancer and healthy cohorts. However, no significant differences were observed between chemotherapy responders and nonresponders within community subgroups. Machine learning models based on responder status outperformed clinical variables in predicting complete response (AUC 0.88 vs. AUC 0.50), although no single microbial species emerged as a fully reliable biomarker.The evaluation of short chain fatty acid (SCFA) concentration in blood and stool revealed no correlation with responder status. Still, SCFA analysis showed a higher abundance of Akkermansia (rs = 0.51, P = 0.017) and Clostridia (rs = 0.52, P = 0.018), which correlated with increased levels of detectable fecal isobutyric acid. Higher levels of fecal Lactobacillus (rs = 0.49, P = 0.02) and Enterobacteriaceae (rs = 0.52, P < 0.03) correlated with increased fecal propionic acid.In conclusion, our study constitutes the first large-scale, multicenter assessment of GM composition, suggesting the potential for a complex microbial signature to predict patients more likely to respond to NAC based on multiple taxa. SIGNIFICANCE: Our study highlights results that link the composition of the GM to the efficacy of NAC in MIBC. We discovered that patients with higher levels of Bacteroides experienced a worse response to NAC. This microbial signature shows promise as a superior predictor of treatment response over traditional clinical variables. Although preliminary, our findings advocate for larger, more detailed studies to validate these associations.

Global Meta-analysis of Urine Microbiome: Colonization of Polycyclic Aromatic Hydrocarbon-degrading Bacteria Among Bladder Cancer Patients.

BACKGROUND: The application of next-generation sequencing techniques has enabled characterization of urinary tract microbiome. Although many studies have demonstrated associations between the human microbiome and bladder cancer (BC), these have not always reported consistent results, thereby necessitating cross-study comparisons. Thus, the fundamental questions remain how we can utilize this knowledge. OBJECTIVE: The aim of our study was to examine the disease-associated changes in urine microbiome communities globally utilizing a machine learning algorithm. DESIGN, SETTING, AND PARTICIPANTS: Raw FASTQ files were downloaded for the three published studies in urinary microbiome in BC patients, in addition to our own prospectively collected cohort. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Demultiplexing and classification were performed using the QIIME 2020.8 platform. De novo operational taxonomic units were clustered using the uCLUST algorithm and defined by 97% sequence similarity and classified at the phylum level against the Silva RNA sequence database. The metadata available from the three studies included were used to evaluate the differential abundance between BC patients and controls via a random-effect meta-analysis using the metagen R function. A machine learning analysis was performed using the SIAMCAT R package. RESULTS AND LIMITATIONS: Our study includes 129 BC urine and 60 healthy control samples across four different countries. We identified a total of 97/548 genera to be differentially abundant in the BC urine microbiome compared with that of healthy patients. Overall, while the differences in diversity metrics were clustered around the country of origin (Kruskal-Wallis, p < 0.001), collection methodology was a driver of microbiome composition. When assessing dataset from China, Hungary, and Croatia, data demonstrated no discrimination capacity to distinguish between BC patients and healthy adults (area under the curve [AUC] 0.577). However, inclusion of samples with catheterized urine improved the diagnostic accuracy of prediction for BC to AUC 0.995, with precision-recall AUC = 0.994. Through elimination of contaminants associated with the collection methodology among all cohorts, our study identified increased abundance of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria Sphingomonas, Acinetobacter, Micrococcus, Pseudomonas, and Ralstonia to be consistently present in BC patients. CONCLUSIONS: The microbiota of the BC population may be a reflection of PAH exposure from smoking, environmental pollutants, and ingestion. Presence of PAHs in the urine of BC patients may allow for a unique metabolic niche and provide necessary metabolic resources where other bacteria are not able to flourish. Furthermore, we found that while compositional differences are associated with geography more than with disease, many are driven by the collection methodology. PATIENT SUMMARY: The goal of our study was to compare the urine microbiome of bladder cancer patients with that of healthy controls and evaluate any potential bacteria that may be more likely to be found in patients with bladder cancer. Our study is unique as it evaluates this across multiple countries, to find a common pattern. After we removed some of the contamination, we were able to localize several key bacteria that are more likely to be found in the urine of bladder cancer patients. These bacteria all share their ability to break down tobacco carcinogens.

Role of Gut Microbiome in Neoadjuvant Chemotherapy Response in Urothelial Carcinoma: A Multi-Institutional Prospective Cohort Evaluation.

Treatment with neoadjuvant chemotherapy (NAC) in muscle invasive bladder cancer (MIBC) is associated with clinical benefit in urothelial carcinoma. While extensive research evaluating role of tumor mutational expression profiles and clinicopathologic factors into chemoresponse has been published, the role of gut microbiome (GM) in bladder cancer in chemoresponse has not been thoroughly evaluated. A working knowledge of the microbiome and its effect on all forms of cancer therapy in BC is critical. Here we examine gut microbiome of bladder cancer patients undergoing NAC. Overall, there was no significant difference in alpha and beta diversity by responder status. However, analysis of fecal microbiome samples showed that a higher abundance of Bacteroides within both institutional cohorts during NAC was associated with residual disease at the time of radical cystectomy regardless of chemotherapy regimen. Group community analysis revealed presence of favorable microbial subtypes in complete responders. Finally, fecal microbial composition outperformed clinical variables in prediction of complete response (AUC 0.88 vs AUC 0.50), however, no single microbial species could be regarded as a fully consistent biomarker. Microbiome-based community signature as compared to single microbial species is more likely to be associated as the link between bacterial composition and NAC response.

Characterization of Changes in Penile Microbiome Following Pediatric Circumcision.

BACKGROUND: While microbiome and host regulation contribute independently to many disease states, it is unclear how circumcision in pediatric population influences subsequent changes in penile microbiome. OBJECTIVE: Our study aims to analyze jointly paired taxonomic profiles and assess pathways implicated in inflammation, barrier protection, and energy metabolism. DESIGN, SETTING, AND PARTICIPANTS: We analyzed 11 paired samples, periurethral collection, before and after circumcision, to generate microbiome and mycobiome profiling. Sample preparation of 16S ribosomal RNA and internal transcribed spacer sequencing was adapted from the methods developed by the National Institutes of Health Human Microbiome Project. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We obtained the predictive functional attributes of the microbial communities between samples using Silva-Tax4Fun and the Greengenes-Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) approach. The predictive functioning of the microbial communities was determined by linearly combining the normalized taxonomic abundances into the precomputed association matrix of Kyoto Encyclopedia of Genes and Genomes orthology reference profiles. RESULTS AND LIMITATIONS: Several notable microbiome and mycobiome compositional differences were observed between pre- and postcircumcision patients. Pairwise comparisons across taxa revealed a significant decrease (p < 0.05, false discovery rate corrected) of microbiome organisms (Clostridiales, Bacteroidales, and Campylobacterales) and mycobiome (Saccharomycetales and Pleosporales) following circumcision. A total of 14 pathways were found to differ in abundance between the pre- and postcircumcision groups (p < 0.005, false discovery rate <0.1 and linear discriminant analysis score >3; five enriched and nine depleted). The pathways reduced after circumcision were mostly involved with amino acid and glucose metabolism, while pathways prior to circumcision were enriched in genetic information processing and transcription processes. As expected, enrichment in methyl-accepting chemotaxis protein, an integral membrane protein involved in directed motility of microbes to chemical cues and environment, occurred prior to circumcision, while the filamentous hemagglutinin pathway (a strong immunogenic protein) was depleted after circumcision CONCLUSIONS: Our results offer greater insight into the host-microbiota relationship of penile circumcision and may serve to lay the groundwork for future studies focused on drivers of inflammation, infection, and oncogenesis. PATIENT SUMMARY: Our study showed a significant reduction in bacteria and fungi after circumcision, particularly anaerobic bacteria, which are known to be potential inducers of inflammation and cancer. This is the first study of its kind showing the changes in microbiome after circumcision, and some of the changes that occur in healthy infants after circumcision that may explain the differences in cancer and inflammatory disorders in adulthood.

Gastrointestinal Microbiome and Mycobiome Changes during Autologous Transplantation for Multiple Myeloma: Results of a Prospective Pilot Study.

Microbiome dysbiosis has been associated with adverse outcomes of hematopoietic cell transplantation (HCT). We hypothesized that exposure to high-dose melphalan and antimicrobials in patients undergoing autologous HCT for plasma cell disorders results in oral and gastrointestinal microbial dysbiosis, which in turn is associated with regimen-related toxicities. We conducted a prospective study describing the longitudinal changes in oral and gastrointestinal bacteriome and mycobiome in this patient population. Our findings show that microbiome composition present at baseline is associated with the incidence and severity of post-transplantation nausea, vomiting, and culture-negative neutropenic fever, as well as with the rate of neutrophil engraftment. We also have evidence of an association between the microbial communities at count nadir and the development of regimen-related gastrointestinal toxicities commonly observed after exposure to high-dose melphalan. Although bacteriome diversity largely recovers within 1 month after transplantation, we observed a continuous decrease in oral and gastrointestinal mycobiome diversity, suggesting that the mycobiome requires a longer time to recover compared with the bacteriome.

Dysbiosis in the oral bacterial and fungal microbiome of HIV-infected subjects is associated with clinical and immunologic variables of HIV infection.

BACKGROUND: The effect of smoking on microbial dysbiosis and the potential consequence of such shift on markers of HIV disease is unknown. Here we assessed the relationship of microbial dysbiosis with smoking and markers of HIV disease. METHODS: Oral wash was collected from: (1) HIV-infected smokers (HIV-SM, n = 48), (2) HIV-infected non-smokers (HIV-NS, n = 24), or (3) HIV-uninfected smokers (UI-SM, n = 24). Microbial DNA was extracted and their bacterial and fungal microbiota (bacteriome and mycobiome, respectively) were characterized using Ion-Torrent sequencing platform. Sequencing data were compared using clustering, diversity, abundance and inter-kingdom correlations analyses. RESULTS: Bacteriome was more widely dispersed than mycobiome, there was no noticeable difference in clustering between groups. Richness of oral bacteriome in HIV-SM was significantly lower than that of UI-SM (P ≤ .03). Diversity of HIV-NS was significantly lower than that of HIV-SM or UI-SM at phylum level (P ≤ .02). Abundance of Phylum Firmicutes was significantly decreased in HIV-NS compared to HIV-SM and UI-SM (P = .007 and .027, respectively), while abundance of Proteobacteria was significantly increased in HIV-NS compared to HIV-SM and UI-SM (P = .0005 and .011, respectively). Fungal phyla did not differ significantly between the three cohorts. Cumulative smoking was positively correlated with Facklamia but negatively with Enhydrobacter, and current alcohol use was negatively correlated with Geniculata. Bacteria Facklamia exhibited weakly positive correlation with longer PI duration (r = 0.094, P = 0.012), and a negative correlation with nadir CD4 count (r = -0.345; P = 0.004), while Granulicatella was negatively correlated with nadir CD4 count (r = -0.329; P = 0.007). Fungus Stemphylium correlated negatively with nadir CD4 (r = -0.323; P = 0.008). CONCLUSIONS: Dysbiosis of the oral microbiota is associated with clinical and immunologic variables in HIV-infected patients.

The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn's Disease-Like Ileitis.

Background: Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice. Methods: Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation. Results: Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice. Conclusions: Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Bacteriome and mycobiome associations in oral tongue cancer.

Squamous cell carcinoma of the oral (mobile) tongue (OMTC), a non-human papilloma virus-associated oral cancer, is rapidly increasing without clear etiology. Poor oral hygiene has been associated with oral cancers, suggesting that oral bacteriome (bacterial community) and mycobiome (fungal community) could play a role. While the bacteriome is increasingly recognized as an active participant in health, the role of the mycobiome has not been studied in OMTC. Tissue DNA was extracted from 39 paired tumor and adjacent normal tissues from patients with OMTC. Microbiome profiling, principal coordinate, and dissimilarity index analyses showed bacterial diversity and richness, and fungal richness, were significantly reduced in tumor tissue (TT) compared to their matched non-tumor tissues (NTT, P<0.006). Firmicutes was the most abundant bacterial phylum, which was significantly increased in TT compared to NTT (48% vs. 40%, respectively; P=0.004). Abundance of Bacteroidetes and Fusobacteria were significantly decreased in TT compared to matched NTT (P≤0.003 for both). Abundance of 22 bacterial and 7 fungal genera was significantly different between the TT and NTT, including Streptococcus, which was the most abundant and significantly increased in the tumor group (34% vs. 22%, P<0.001). Abundance of fungal genus Aspergillus in TT correlated negatively with bacteria (Actinomyces, Prevotella, Streptococcus), but positively with Aggregatibacter. Patients with high T-stage disease had lower mean differences between TT and NTT compared with patients with low T-stage disease (0.07 vs. 0.21, P=0.04). Our results demonstrate differences in bacteriome and mycobiome between OMTC and their matched normal oral epithelium, and their association with T-stage.

Metabolomic analysis identifies differentially produced oral metabolites, including the oncometabolite 2-hydroxyglutarate, in patients with head and neck squamous cell carcinoma.

BACKGROUND: Metabolomics represents a promising approach for discovering novel targets and biomarkers in head and neck squamous cell carcinoma (HNSCC). Here we used metabolomics to identify oral metabolites associated with HNSCC. METHODS: Tumor and adjacent normal tissue from surgical resections and presurgical oral washes as well as oral washes were collected from healthy participants. Metabolites extractions of these samples were analyzed by liquid chromatography-mass spectroscopy (LC/MS), LC/MS/MS and gas chromatography-MS (GC/MS). RESULTS: Among 28 samples obtained from 7 HNSCC cases and 7 controls, 422 metabolites were detected (269 identified and 153 unidentified). Oral washes contained 12 and 23 metabolites in healthy controls and HNSCC patients, respectively, with phosphate and lactate being the most abundant. Small molecules related to energy metabolism were significantly elevated in HNSCC patients compared to controls. Levels of beta-alanine, alpha-hydroxyisovalerate, tryptophan, and hexanoylcarnitine were elevated in HNSCC oral washes compared to healthy controls (range 7.8-12.2-fold). Resection tissues contained 22 metabolites, of which eight were overproduced in tumor by 1.9- to 12-fold compared to controls. TCA cycle analogs 2-hydroxyglutarate (2-HG) and 3-GMP were detected exclusively in tumor tissues. Targeted quantification of 2-HG in a representative HNSCC patient showed increase in tumor tissue (14.7 µg/mL), but undetectable in normal tissue. Moreover, high levels of 2-HG were detected in HNSCC cell lines but not in healthy primary oral keratinocyte cultures. CONCLUSIONS: Oral metabolites related to energy metabolism were elevated in HNSCC, and acylcarnitine and 2HG may have potential as non-invasive biomarkers. Further validation in clinical studies is warranted.

Clinical Effects of Gamma-Radiation-Resistant Aspergillus sydowii on Germ-Free Mice Immunologically Prone to Inflammatory Bowel Disease.

We report and investigated a case of inadvertent contamination of 125 mice (housed in two germ-free positive-pressurized isolators) with emerging human and coral pathogen Aspergillus sydowii. The infected mice correspond to genetic line SAMP1/YitFc, which have 100% immune predisposition to develop Crohn's disease-like spontaneous pathologies, namely, inflammatory bowel disease (IBD). Pathogen update based on a scoping review of the literature and our clinical observations and experimentation are discussed. The unwanted infection of germ-free mice (immunologically prone to suffer chronic inflammation) with human pathogen A. sydowii resulted in no overt signs of clinical disease over 3-week exposure period, or during DSS-induced colitis experiments. Results and observations suggest that A. sydowii alone has limited clinical effect in immunocompromised germ-free mice or that other commensal microbial flora is required for Aspergillus-associated disease to occur.

The Fungal Biome of the Oral Cavity.

Organisms residing in the oral cavity (oral microbiota) contribute to health and disease, and influence diseases like gingivitis, periodontitis, and oral candidiasis (the most common oral complication of HIV-infection). These organisms are also associated with cancer and other systemic diseases including upper respiratory infections. There is limited knowledge regarding how oral microbes interact together and influence the host immune system. Characterizing the oral microbial community (oral microbiota) in health and disease represents a critical step in gaining insight into various members of this community. While most of the studies characterizing oral microbiota have focused on bacterial community, there are few encouraging studies characterizing the oral mycobiome (the fungal component of the oral microbiota). Our group recently characterized the oral mycobiome in health and disease focusing on HIV. In this chapter we will describe the methods used by our group for characterization of the oral mycobiome.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 µM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

Angiogenic pretreatment to enhance myocardial function after cellular cardiomyoplasty with skeletal myoblasts.

OBJECTIVE: Improvements in ventricular function after cellular cardiomyoplasty appear to be limited by the poor survival of the cellular implants. Angiogenic pretreatment of infarcted myocardium may improve implanted cell survival and consequently myocardial function. METHODS: Fischer 344 rats underwent coronary artery ligation and injection of an adenovirus encoding vascular endothelial growth factor 121 or of saline solution at increasing intervals after ligation. Myocardial perfusion and mass preservation were assessed. On the basis of these data, four groups of animals underwent coronary ligation and adenovirus with or without syngeneic skeletal myoblast administration: (1) adenovirus at ligation and myoblasts 3 weeks later (n = 7), (2) saline solution at ligation and myoblasts 3 weeks later (n = 8), (3) saline solution at ligation and 3 weeks later (n = 8), and (4) saline solution at ligation and adenovirus with myoblasts 3 weeks later (n = 5). Left ventricular ejection fraction was analyzed by echocardiography before coronary ligation and 3 and 5 weeks later, after which cell survival was assessed in harvested tissues. RESULTS: Myocardial infarct perfusion was at least 50% greater in animals treated with adenoviral vector than with saline solution immediately after ligation (P < .02). In comparison, delayed adenovirus administration did not significantly diminish infarct perfusion but resulted in decreased myocardial preservation (P < .05). Accordingly, adenovirus administration nearly tripled implanted myoblast survival relative to saline solution-treated animals (P = .004). Left ventricular ejection fraction was improved, however, only after cell implantation with adenovirus pretreatment (P = .027). CONCLUSION: Angiogenic strategies can help to preserve myocardium jeopardized by acute coronary occlusions. Angiogenic pretreatment enhances the efficacy of cellular cardiomyoplasty.

An Egr-1 master switch for arteriogenesis: studies in Egr-1 homozygous negative and wild-type animals.

BACKGROUND: Arteriogenesis has been implicated as an important biologic response to acute vascular occlusion. The early growth response 1 (Egr-1) gene encodes an immediate-early response transcription factor that is upregulated by changes in vascular strain and that in turn upregulates a number of putative angiogenic and arteriogenic growth factors. We therefore hypothesized that early growth response 1 might be a critical arteriogenic messenger that induces revascularization in the setting of acute vascular occlusions. METHODS: Wild-type or Egr-1-/- (null) C57 BL mice, or Sprague-Dawley rats, underwent unilateral iliofemoral artery excision and subsequent analyses for angiogenesis and arteriogenesis through cell-specific immunohistochemistry. Rats were also administered an adenoviral vector encoding for Egr-1 (AdEgr group), noncoding vectors (AdNull group), or saline, after which these animals were assessed by means of serial laser Doppler perfusion imaging and morphologic examination of rat foot-pad ischemic lesions. RESULTS: Egr-1 wild-type mice demonstrated an equivalent number of capillaries but a greater number of arterioles following excision versus Egr-1 null mice. AdEgr group rats demonstrated greater distal perfusion from 7 to 21 days after excision compared with control animals (P < .02), which approximated normal perfusion at 21 days after excision. AdEgr group rats also demonstrated greater arteriolar density and less severe ischemic foot-pad lesions than control animals. CONCLUSION: These data suggest the importance of Egr-1 as a critical and potentially therapeutic mediator of revascularization after vascular occlusion and implicate arteriogenesis (collateral vessel formation) as a critical component of this process.

Homozygous deletion of early growth response 1 gene and critical limb ischemia after vascular ligation in mice: evidence for a central role in vascular homeostasis.

BACKGROUND: The early growth response 1 gene (Egr1) encodes for an immediate to early response transcription factor that is upregulated by changes in vascular strain and hypoxia and in turn upregulates the downstream expressions of a number of angiogenic growth factors. We therefore hypothesized that early growth response 1 may be a critical early messenger governing revascularization in the setting of acute vascular occlusions. METHODS: C57 BL/6 mice deficient in the Egr1 gene (knockout) and their wild-type litter mates underwent ligation and excision of the femoral artery with or without the previous administration of 2.7 x 10(9) particle units of an adenoviral vector coding for the vascular endothelial growth factor gene (VEGF) or Egr1. Distal hind limb perfusion was serially measured in these animals with laser Doppler perfusion imaging. RESULTS: Wild-type mice (n = 9) had nearly complete restitution of hind limb perfusion by day 35 after ligation. In contrast, all noninjected Egr1 knockout mice (n = 5) had severe ipsilateral limb necrosis develop between 1 and 4 days after ligation (P <.0001). Egr1 knockout mice injected with VEGF vector (n = 4) demonstrated significantly improved perfusion relative to baseline by postligation day 28, which persisted to postligation day 35 (P <.05). Egr1 knockout animals injected with Egr1 vector (n = 7) demonstrated a partial recovery of hind limb perfusion relative to VEGF vector-treated knockout animals at postligation day 4 (P <.01), which persisted to day 35. CONCLUSIONS: These findings suggest that early growth response 1 plays a pivotal role in reperfusion responses to vascular occlusion in mice and possibly other mammals.

Angiogenic pretreatment improves the efficacy of cellular cardiomyoplasty performed with fetal cardiomyocyte implantation.

BACKGROUND: Cell implantation into areas of myocardial infarction (cellular cardiomyoplasty) may be limited in efficacy because of the lack of blood supply to these areas of myocardium, resulting in early loss of transplanted cells. We therefore tested the hypothesis that pretreatment of infarcted myocardium with angiogenic therapy, followed by cell transplant, would be more effective than the application of either strategy alone. METHODS: Fischer 344 rats underwent left coronary artery ligation and injection of an adenovirus encoding VEGF 121, an empty expression cassette control vector, or saline solution. Capillary density in the infarcted region was determined in preliminary studies. Cardiomyocytes harvested from syngeneic Fischer rat fetuses were prelabeled and then injected directly into the infarct area 3 weeks after vector administration. Exercise treadmill testing was performed 2 weeks after cell transplantation, after which a cell viability index was calculated as the number of implanted (prelabeled) nuclei divided by the number of coadministered microspheres detected in sections of implanted myocardium. RESULTS: Capillary density in the area of infarction was significantly greater in adenovirus encoding VEGF 121 compared with rats injected with saline solution (P =.001). The cell survival index was also greater in adenovirus encoding VEGF 121 compared with animals injected with empty expression cassette control or saline solution (P =.0045). Exercise tolerance was nearly doubled in animals receiving adenovirus encoding VEGF 121 3 weeks prior to cell implantation compared with animals receiving adenovirus encoding VEGF 121 or cells alone or those receiving adenovirus encoding VEGF 121 at the time of cell implantation (P <.001). CONCLUSIONS: Pretreatment of an infarcted region of the heart with angiogenic mediators such as VEGF can enhance the efficacy of cellular cardiomyoplasty, presumably by creating a more favorable environment for the survival of transplanted cells.

Adenoviral-mediated transfer of vascular endothelial growth factor 121 cDNA enhances myocardial perfusion and exercise performance in the nonischemic state.

BACKGROUND: Angiogenic gene therapy has been demonstrated to enhance perfusion to ischemic tissues, but it is unknown whether the administration of angiogenic growth factors will increase blood flow to nonischemic tissues. This study investigates whether enhanced myocardial perfusion can be mediated by adenovirus-mediated transfer of vascular endothelial growth factor 121 cDNA to nonischemic myocardium. METHODS: New Zealand White rabbits received adenovirus (5 x 10(10) particle units) encoding for vascular endothelial growth factor 121 (n = 14) or a control vector without a transgene (n = 13) or saline solution (n = 9) via direct myocardial injection. Fluorescent microsphere perfusion studies and histologic analyses were performed 4 weeks later. In a parallel study, exercise treadmill testing was performed to assess the functional effects of this therapy in Sprague-Dawley rats. RESULTS: Microsphere assessment of myocardial perfusion in rabbits 4 weeks after adenovirus-encoding vascular endothelial growth factor administration was greater than that for rats injected with control vector without a transgene or saline solution (3.2 +/- 0.5 vs 2.7 +/- 0.7 and 2.4 +/- 0.4, respectively; P <.03). The endothelial cell count per high power field was increased in animals injected with adenovirus-encoding vascular endothelial growth factor versus animals injected with control vector without a transgene or saline solution (147 +/- 27 vs 123 +/- 14 and 125 +/- 16 cells, respectively), although this did not reach statistical significance (P =.12). Rats treated with adenovirus-encoding vascular endothelial growth factor also demonstrated prolonged exercise tolerance compared with rats injected with control vector without a transgene or saline solution (exhaustion time: 26 +/- 5 minutes vs 19 +/- 2 minutes and 20 +/- 3 minutes, respectively; P =.006). CONCLUSIONS: Adenovirus encoding-mediated transfer of vascular endothelial growth factor 121 induces an enhancement in regional perfusion in nonischemic myocardium that corresponds to changes in exercise tolerance. Adenovirus-encoding vascular endothelial growth factor therapy may be useful for inducing angiogenesis in the nonischemic state, such as for prophylactic therapy of early coronary artery disease.