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The use of high flow nasal cannula (HFNC) has become widely used in pediatric intensive care units (PICUs) throughout the world. The rapid adoption has outpaced the number of studies evaluating the safety and efficacy in a variety of pediatric diseases/conditions.This scoping review begins with the definition and mechanisms of action of HFNC and then follows with a review of the literature focused on studies performed on critically ill children cared for in the PICU. The PubMed database was searched with a pediatric filter from the time period 2000 to 2021.The rapid adoption of HFNC in PICUs has largely been driven by changes in institutional practices and small observational studies. There is a lack of adequately powered studies evaluating patient-centered outcomes, such as intubation rates, mortality, PICU, and hospital length of stay. Given the wide variability in flow rates and clinical indications, more research is needed to better define effective flow rates for different disease states as well as markers of treatment success and failure. One particular entity that is poorly studied is the use of HFNC in those at risk for developing pediatric acute respiratory distress syndrome (PARDS).

Objectives To derive and internally validate a bronchiolitis-specific illness severity score (the Critical Bronchiolitis Score) that out-performs mortality-based illness severity scores (e.g., Pediatric Risk of Mortality) in measuring expected duration of respiratory support and PICU length of stay for critically ill children with bronchiolitis. Design Retrospective database study using the Virtual Pediatric Systems (VPS, LLC; Los Angeles, CA) database. Setting One-hundred twenty-eight North-American PICUs. Patients Fourteen-thousand four-hundred seven children less than 2 years old admitted to a contributing PICU with primary diagnosis of bronchiolitis and use of ICU-level respiratory support (defined as high-flow nasal cannula, noninvasive ventilation, invasive mechanical ventilation, or negative pressure ventilation) at 12 hours after PICU admission. Interventions Patient-level variables available at 12 hours from PICU admission, duration of ICU-level respiratory support, and PICU length of stay data were extracted for analysis. After randomly dividing the cohort into derivation and validation groups, patient-level variables that were significantly associated with the study outcomes were selected in a stepwise backward fashion for inclusion in the final score. Score performance in the validation cohort was assessed using root mean squared error and mean absolute error, and performance was compared with that of existing PICU illness severity scores. Measurements and main results Twelve commonly available patient-level variables were included in the Critical Bronchiolitis Score. Outcomes calculated with the score were similar to actual outcomes in the validation cohort. The Critical Bronchiolitis Score demonstrated a statistically significantly stronger association with duration of ICU-level respiratory support and PICU length of stay than mortality-based scores as measured by root mean squared error and mean absolute error. Conclusions The Critical Bronchiolitis Score performed better than PICU mortality-based scores in measuring expected duration of ICU-level respiratory support and ICU length of stay. This score may have utility to enrich interventional trials and adjust for illness severity in observational studies in this very common PICU condition.

Background:Infants asymptomatically excrete Clostridioides difficile during their first year of life, suggesting that they may represent a source of infection for adults who acquire community-associated C. difficile infection (CA-CDI). The genetic relationship of C. difficile strains from asymptomatic infants and adults with CA-CDI is not well defined.Methods:In this study, 50 infants were recruited at birth, and stool samples were collected at routine well-child visits. Adult stool samples collected during the same period and geographical area from patients who were diagnosed with CA-CDI were selected for comparison. C. difficile was cultivated and probed by PCR for toxin genes and were typed by PCR fluorescent ribotyping. Isolates from adults and infants with shared ribotypes were subjected to whole-genome sequencing (WGS).Results:Of these 50 infants, 36 were positive for C. difficile at least once in their first year of life, with a peak incidence at 6 months. Among 180 infant stool samples, 48 were positive. Of 48 isolates from positive stools, 29 were toxigenic by polymerase chain reaction (PCR) and 8 of 48 stool samples were positive for toxin by enzyme immunoassays (EIAs). Ribotypes F106 and F014-020 were present in both colonized infants and adults with CA-CDI. WGS identified 1 adult–infant pair that differed by 5 single-nucleotide polymorphisms (SNPs). Also, 4 additional adult–infant clusters differed by ≤16 SNPs.Conclusions:Infants that are colonized with C. difficile share ribotypes with adults from the same geographical region with CA-CDI. Selected isolates in the 2 populations show a genetic relationship by WGS.

Blood culture overuse in the pediatric intensive care unit (PICU) can lead to unnecessary antibiotic use and contribute to antibiotic resistance. Optimizing blood culture practices through diagnostic stewardship may reduce unnecessary blood cultures and antibiotics.To evaluate the association of a 14-site multidisciplinary PICU blood culture collaborative with culture rates, antibiotic use, and patient outcomes.This prospective quality improvement (QI) collaborative involved 14 PICUs across the United States from 2017 to 2020 for the Bright STAR (Testing Stewardship for Antibiotic Reduction) collaborative. Data were collected from each participating PICU and from the Children's Hospital Association Pediatric Health Information System for prespecified primary and secondary outcomes.A local QI program focusing on blood culture practices in the PICU (facilitated by a larger QI collaborative).The primary outcome was blood culture rates (per 1000 patient-days/mo). Secondary outcomes included broad-spectrum antibiotic use (total days of therapy and new initiations of broad-spectrum antibiotics ≥3 days after PICU admission) and PICU rates of central line-associated bloodstream infection (CLABSI), Clostridioides difficile infection, mortality, readmission, length of stay, sepsis, and severe sepsis/septic shock.Across the 14 PICUs, the blood culture rate was 149.4 per 1000 patient-days/mo preimplementation and 100.5 per 1000 patient-days/mo postimplementation, for a 33% relative reduction (95% CI, 26%-39%). Comparing the periods before and after implementation, the rate of broad-spectrum antibiotic use decreased from 506 days to 440 days per 1000 patient-days/mo, respectively, a 13% relative reduction (95% CI, 7%-19%). The broad-spectrum antibiotic initiation rate decreased from 58.1 to 53.6 initiations/1000 patient-days/mo, an 8% relative reduction (95% CI, 4%-11%). Rates of CLABSI decreased from 1.8 to 1.1 per 1000 central venous line days/mo, a 36% relative reduction (95% CI, 20%-49%). Mortality, length of stay, readmission, sepsis, and severe sepsis/septic shock were similar before and after implementation.Multidisciplinary diagnostic stewardship interventions can reduce blood culture and antibiotic use in the PICU. Future work will determine optimal strategies for wider-scale dissemination of diagnostic stewardship in this setting while monitoring patient safety and balancing measures.

Objectives Negative pressure ventilation may be more physiologic than positive pressure ventilation, but data describing negative pressure ventilation use in the PICU are limited. We aimed to describe the epidemiology and outcomes of PICU patients receiving negative pressure ventilation. Design Descriptive analysis of a large, quality-controlled multicenter database. Setting Fifty-six PICUs in the Virtual Pediatric Systems database who reported use of negative pressure ventilation. Patients Children admitted to a participating PICU between 2009 and 2019 who received negative pressure ventilation. Interventions None. Measurements and main results Among 788 subjects, 71% were less than 2 years old, and 45% had underlying health conditions. Two concurrent aspiration events were the only adverse events reported. After excluding one over-represented center, the 3 years with the most negative pressure ventilation usage were 2017-2019 (all > 25 cases/yr and ≥ 13 centers reporting usage). Among those 187 children, the most common primary diagnoses were bronchiolitis and cardiac disease (both 15.5%), 24.1% required endotracheal intubation after negative pressure ventilation, and 9.1% died. Conclusions Negative pressure ventilation is being used in many PICUs, most commonly for pulmonary infections or cardiac disease, in children with high rates of subsequent intubation and mortality and with few documented adverse events. Use at individual centers is rare but increasing, suggesting need for prospective collaboration to better evaluate the role of negative pressure ventilation in the PICU.

Infants asymptomatically excrete Clostridioides difficile during their first year of life, suggesting that they may represent a source of infection for adults who acquire community-associated C. difficile infection (CA-CDI). The genetic relationship of C. difficile strains from asymptomatic infants and adults with CA-CDI is not well defined.In this study, 50 infants were recruited at birth, and stool samples were collected at routine well-child visits. Adult stool samples collected during the same period and geographical area from patients who were diagnosed with CA-CDI were selected for comparison. C. difficile was cultivated and probed by PCR for toxin genes and were typed by PCR fluorescent ribotyping. Isolates from adults and infants with shared ribotypes were subjected to whole-genome sequencing (WGS).Of these 50 infants, 36 were positive for C. difficile at least once in their first year of life, with a peak incidence at 6 months. Among 180 infant stool samples, 48 were positive. Of 48 isolates from positive stools, 29 were toxigenic by polymerase chain reaction (PCR) and 8 of 48 stool samples were positive for toxin by enzyme immunoassays (EIAs). Ribotypes F106 and F014-020 were present in both colonized infants and adults with CA-CDI. WGS identified 1 adult-infant pair that differed by 5 single-nucleotide polymorphisms (SNPs). Also, 4 additional adult-infant clusters differed by ≤16 SNPs.Infants that are colonized with C. difficile share ribotypes with adults from the same geographical region with CA-CDI. Selected isolates in the 2 populations show a genetic relationship by WGS.

Objectives High-flow nasal cannula and noninvasive positive pressure ventilation are used to support children following liberation from invasive mechanical ventilation. Evidence comparing extubation failure rates between patients randomized to high-flow nasal cannula and noninvasive positive pressure ventilation is available for adult and neonatal patients; however, similar pediatric trials are lacking. In this study, we employed a quality controlled, multicenter PICU database to test the hypothesis that high-flow nasal cannula is associated with higher prevalence of reintubation within 24 hours among patients with bronchiolitis. Design Secondary analysis of a prior study utilizing the Virtual Pediatric Systems database. Setting One-hundred twenty-four participating PICUs. Patients Children less than 24 months old with a primary diagnosis of bronchiolitis who were admitted to one of 124 PICUs between January 2009 and September 2015 and received invasive mechanical ventilation. Interventions None. Measurements and main results Among 759 patients, median age was 2.4 months (1.3-5.4 mo), 41.2% were female, 39.7% had greater than or equal to 1 comorbid condition, and 43.7% were Caucasian. Median PICU length of stay was 8.7 days (interquartile range, 5.8-13.7 d) and survival to PICU discharge was 100%. Median duration of intubation was 5.5 days (3.4-9.0 d) prior to initial extubation. High-flow nasal cannula was used following extubation in most (656 [86.5%]) analyzed subjects. The overall prevalence of reintubation within 24 hours was 5.9% (45 children). Extubation to noninvasive positive pressure ventilation was associated with greater prevalence of reintubation than extubation to high-flow nasal cannula (11.7% vs 5.0%; p = 0.016) and, in an a posteriori model that included Pediatric Index of Mortality 2 score and comorbidities, was associated with increased odds of reintubation (odds ratio, 2.43; 1.11-5.34; p = 0.027). Conclusions In this secondary analysis of a multicenter database of children with bronchiolitis, extubation to high-flow nasal cannula was associated with a lower prevalence of reintubation within 24 hours compared with noninvasive positive pressure ventilation in both unmatched and propensity-matched analysis. Prospective trials are needed to determine if post-extubation support modality can mitigate the risk of extubation failure.

Neurologic and functional morbidity occurs in ~30% of PICU survivors, and young children may be at particular risk. Bronchiolitis is a common indication for PICU admission among children less than 2 years old. Two single-center studies suggest that greater than 10-25% of critical bronchiolitis survivors have neurologic and functional morbidity but those estimates are 20 years old. We aimed to estimate the burden of neurologic and functional morbidity among more recent bronchiolitis patients using two large, multicenter databases.Analysis of the Pediatric Health Information System and the Virtual Pediatric databases.Forty-eight U.S. children's hospitals (Pediatric Health Information System) and 40 international (mostly United States) children's hospitals (Virtual Pediatric Systems).Previously healthy PICU patients less than 2 years old admitted with bronchiolitis between 2009 and 2015 who survived and did not require extracorporeal membrane oxygenation or cardiopulmonary resuscitation.None. Neurologic and functional morbidity was defined as a Pediatric Overall Performance Category greater than 1 at PICU discharge (Virtual Pediatric Systems subjects), or a subsequent hospital encounter involving developmental delay, feeding tubes, MRI of the brain, neurologist evaluation, or rehabilitation services (Pediatric Health Information System subjects).Among 3,751 Virtual Pediatric Systems subjects and 9,516 Pediatric Health Information System subjects, ~20% of patients received mechanical ventilation. Evidence of neurologic and functional morbidity was present at PICU discharge in 707 Virtual Pediatric Systems subjects (18.6%) and more chronically in 1,104 Pediatric Health Information System subjects (11.6%). In both cohorts, neurologic and functional morbidity was more common in subjects receiving mechanical ventilation (27.5% vs 16.5% in Virtual Pediatric Systems; 14.5% vs 11.1% in Pediatric Health Information System; both p0.001). In multivariate models also including demographics, use of mechanical ventilation was the only variable that was associated with increased neurologic and functional morbidity in both cohorts.In two large, multicenter databases, neurologic and functional morbidity was common among previously healthy children admitted to the PICU with bronchiolitis. Prospective studies are needed to measure neurologic and functional outcomes using more precise metrics. Identification of modifiable risk factors may subsequently lead to improved outcomes from this common PICU condition.

OBJECTIVES Initial respiratory support with noninvasive positive pressure ventilation or high-flow nasal cannula may prevent the need for invasive mechanical ventilation in PICU patients with bronchiolitis. However, it is not clear whether the initial choice of respiratory support modality influences the need for subsequent invasive mechanical ventilation. The purpose of this study is to compare the rate of subsequent invasive mechanical ventilation after initial support with noninvasive positive pressure ventilation or high-flow nasal cannula in children with bronchiolitis. DESIGN Analysis of the Virtual Pediatric Systems database. SETTING Ninety-two participating PICUs. PATIENTS Children less than 2 years old admitted to a participating PICU between 2009 and 2015 with a diagnosis of bronchiolitis who were prescribed high-flow nasal cannula or noninvasive positive pressure ventilation as the initial respiratory treatment modality. INTERVENTIONS None. Subsequent receipt of invasive mechanical ventilation was the primary outcome. MEASUREMENTS AND MAIN RESULTS We identified 6,496 subjects with a median age 3.9 months (1.7-9.5 mo). Most (59.7%) were male, and 23.4% had an identified comorbidity. After initial support with noninvasive positive pressure ventilation or high-flow nasal cannula, 12.3% of patients subsequently received invasive mechanical ventilation. Invasive mechanical ventilation was more common in patients initially supported with noninvasive positive pressure ventilation compared with high-flow nasal cannula (20.1% vs 11.0%: p < 0.001). In a multivariate logistic regression model that adjusted for age, weight, race, viral etiology, presence of a comorbid diagnosis, and Pediatric Index of Mortality score, initial support with noninvasive positive pressure ventilation was associated with a higher odds of subsequent invasive mechanical ventilation compared with high-flow nasal cannula (odds ratio, 1.53; 95% CI, 1.24-1.88). CONCLUSIONS In this large, multicenter database study of infants with acute bronchiolitis that received initial respiratory support with high-flow nasal cannula or noninvasive positive pressure ventilation, noninvasive positive pressure ventilation use was associated with higher rates of invasive mechanical ventilation, even after adjusting for demographics, comorbid condition, and severity of illness. A large, prospective, multicenter trial is needed to confirm these findings.

We focus on two recent aspects of Clostridium difficile infection (CDI) in children, namely the emergence of community-associated CDI (CA-CDI) and the incidence and prevention of recurrent CDI. Current surveys suggest that a large proportion of all pediatric CDI is acquired in the community. Risk factors and frequency estimates of pediatric CA-CDI, however, are confounded in babies and toddlers by a high rate of asymptomatic excretion, whose detection likely is exaggerated by the wide use of highly sensitive nucleic acid amplification tests. Recurrent diarrhea occurs in up to 25% of children with CDI. Preventative strategies for recurrent CDI in adults, namely pulse and taper antibiotic dosing, use of anti-CDI drugs with mild effect on the colonic microbiome, fecal microbiota transplantation, and passive immune therapy, currently are being tested in children. Future studies are required to better characterize community acquisition of CDI in children and to define the safety and effectiveness of preventative strategies for recurrent CDI.

Background The number of adults afflicted with community-associated Clostridium difficile infection (CA-CDI) has increased dramatically over the past 10 years. Exposure to infants, a population known to be asymptomatically colonized by C. difficile (CD), has been identified as a risk factor for CA-CDI, implying that infants may be a reservoir for adult infection. In the present study, we determined the distribution of CD ribotypes isolated from adults with CA-CDI and compared them to the ribotypes of strains excreted asymptomatically among a cohort of healthy infants from the same geographical location. Methods Adult samples submitted to a referral university hospital microbiology laboratory as part of routine care were identified as CD+ by commercial PCR and stored at −80°; the subset of samples from patients meeting IDSA criteria for CA-CDI were selected for further analysis. A cohort of healthy infants attending a suburban, demographically diverse pediatric practice 6 miles from the hospital were enrolled at birth and prospectively followed at 2-, 6-, and 12-month well-visits. Stool collected at each infant visit was cultivated for CD using routine techniques. DNA from both sets of organisms was extracted and subjected to fluorescent PCR ribotyping. Amplicons were assigned to specific ribotypes through sequence analysis, using the nomenclature proposed previously (J Clin Microbiol 2015;53:1192). Results To date, 29 adult samples (collected between August 1, 2016 and January 31, 2018) and 32 infant samples (collected between July 1, 2016 and March 31, 2018) have been ribotyped. Eleven (18%) organisms could not be typed (3 adult; 8 infant). The most representative ribotype identified in the adult CA-CDI samples was F014-020 (54%), with small numbers scattered among six other ribotypes. The most prominent ribotypes in infants were F106 (33%), F010 (17%), and F012 (17%); two (8%) infants were colonized with ribotype F014-020. Except for F014-020, there was no concordance of ribotypes among adult CA-CDI and infant isolates. Conclusion In this population, a small proportion of asymptomatic infants were colonized by a prominent CA-CDI ribotype in adults, but other ribotypes were unique to each age group. Disclosures All authors: No reported disclosures.

Background Recent years have witnessed an explosive increase in community-associated Clostridium difficile infection (CA-CDI) in adults. Contact with infants, a population known to be asymptomatically colonized by C. difficile (CD), has been identified as a risk factor for CA-CDI, rendering it vital to explore the epidemiology and determinants of acquisition in babies. Methods In this prospective cohort study, healthy infants attending a demographically diverse suburban pediatric practice were enrolled at birth and followed through their 2-month, 6-month, and 12-month well child visit. At each visit, stool samples were collected, and questionnaires including interim exposure to potential risk factors for CD acquisition were administered. Stool was inoculated on pre-reduced CCFA agar with a graduated loop. Among CD isolates, toxin genes were identified by PCR. Results Fifty infants were recruited; 90% of samples and questionnaires were completed. The average gestational age was 39 weeks and 46% were male. Twenty-eight (56%) infants had at least one sample positive for CD during the study: cross sectional incidence was 0/50 at birth; 9/47 (19%) at 2 months; 22/43 (51%) at 6 months; 6/37 (16%) at 1 year. Of those with positive stool cultures, three(11%) tested positive at multiple visits. Of the 37 (81%) isolates, 30 were PCR-positive for CD toxin. Five stool samples harbored >4.5 log10 cfu of toxigenic CD/g of stool. Proportions of CD+ vs. CD− subjects, respectively, with interim exposure to selected CD risk factors at each visit were as follows: infant healthcare visit 45% vs. 42%; household member healthcare visit 17% vs. 23%; household member with diarrhea 14% vs. 29%; antibiotic exposure 5% vs. 4%; antacid exposure 7% vs. 3%, all P > 0.05. Regarding risks for acquisition of enteric pathogens in general: breastmilk-including nutrition 57% vs. 73% (P < 0.05 only at 2-month visit); 48% CD+ infants had interim daycare attendance vs. 25% CD− (but P > 0.05 at each visit). Conclusion Asymptomatic carriage of toxigenic CD occurred in over half of healthy infants during the first year of life, and several had a high organism burden that could increase the risk for transmission. While daycare attendance was more common among colonized infants, the majority of infants who were CD+ had no daycare exposure. Disclosures All authors: No reported disclosures.

Catecholamine stimulation of α1-adrenoceptors exerts growth factor-like activity, mediated by generation of reactive oxygen species, on arterial smooth muscle cells and adventitial fibroblasts and contributes to hypertrophy and hyperplasia in models of vascular injury and disease. Adrenergic trophic activity also contributes to flow-mediated positive arterial remodeling by augmenting proliferation and leukocyte accumulation. To further examine this concept, we studied whether catecholamines contribute to collateral growth and angiogenesis in hindlimb insufficiency. Support for this hypothesis includes the above-mentioned studies, evidence that ischemia augments norepinephrine release from sympathetic nerves, and proposed involvement of reactive oxygen species in angiogenesis and collateral growth. Mice deficient in catecholamine synthesis [by gene deletion of dopamine β-hydroxylase (DBH−/−)] were studied. At 3 wk after femoral artery ligation, increases in adductor muscle perfusion were similar in DBH−/−and wild-type mice, whereas recovery of plantar perfusion and calf microsphere flow were attenuated, although not significantly. Preexisting collaterals in adductor of wild-type mice showed increases in lumen diameter (60%) and medial and adventitial thickness (57 and 119%, P < 0.05 here and below). Lumen diameter increased similarly in DBH−/−mice (52%); however, increases in medial and adventitial thicknesses were reduced (30 and 65%). Leukocyte accumulation in the adventitia/periadventitia of collaterals was 39% less in DBH−/−mice. Increased density of α-smooth muscle actin-positive vessels in wild-type adductor (45%) was inhibited in DBH−/−mice (2%). Although both groups experienced similar atrophy in the gastrocnemius (∼22%), the increase in capillary-to-muscle fiber ratio in wild-type mice (21%) was inhibited in DBH−/−mice (7%). These data suggest that catecholamines may contribute to collateral growth and angiogenesis in tissue ischemia.

Using sequences from the plastid trnL-F region and nrDNA ITS, we investigated the phylogeny of the fleshy-fruited African tribe Haemantheae of the Amaryllidaceae across 19 species representing all genera of the tribe. ITS and a combined matrix produce the most resolute and well-supported tree with parsimony analysis. Two main clades are resolved, one comprising the monophyletic rhizomatous genera Clivia and Cryptostephanus, and a larger clade that unites Haemanthus and Scadoxus as sister genera to an Apodolirion/Gethyllis subclade. One of four included Gethyllis species, G. lanuginosa, resolves as sister to Apodolirion with ITS. Relationships among the Clivia species are not in agreement with a previous published phylogeny. Biogeographic analysis using the divergence/vicariance method roots the tribe in Eastern South Africa, with several subsequent dispersals to the winter rainfall Western Cape region. Chromosomal change from an ancestral 2n=22 (characteristic of Clivia) is associated with each main clade. Reduction in number has occurred in all but Cryptostephanus, which has 2n=24 chromosomes. Increasing the sampling across all of the species in the tribe will allow a more detailed understanding of the biogeographic patterns inherent in the parsimony topology, which undoubtedly reflect Quaternary climatic changes in Southern Africa.

The genus Crinum L. is the only pantropical genus of the Amaryllidaceae. Phylogenetic and biogeographical analyses of nrDNA ITS and plastid trnL-F sequences for all continental groups of the genus Crinum and related African genera are presented, with the genus Amaryllis used as outgroup. ITS indicates that C. baumii is more closely related to Ammocharis and Cybistetes than to Crinum sensu stricto. Three clades are resolved in Crinum s.s. One unites a monophyletic American group with tropical and North African species. The second includes all southern African species and the Australian endemic C. flaccidum. The third includes monophyletic Madagascar, Australasian and Sino-Himalayan clades, with southern African species. The trnL-F phylogeny resolves an American and an Asian/Madagscar clade, and confirms the relationship of C. flaccidum with species endemic to southern Africa. The salverform, actinomorphic perianths of subg. Crinum appear to have evolved several times in the genus from ancestors with zygomorphic perianths (subg. Codonocrinum), thus neither subgenus is monophyletic. Biogeographical analyses place the origin of Crinum in southern Africa, as the region is optimized at all ancestral nodes in the tree topology, and in basal interior nodes of all but one of the major clades. The genus underwent three major waves of radiation corresponding to the three main clades resolved in our trees. Two entries into Australia for the genus are indicated, as are separate Sino-Himalayan and Australasian dispersal events. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363.

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1 +/− ), VEGF receptor-2 (VEGFR-2 +/− ), and overexpressing (VEGF hi/+ ) and underexpressing VEGF-A (VEGF lo/+ ). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P +/− showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1 +/− showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGF lo/+ had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGF hi/+ mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGF hi/+ mice, VEGF lo/+ formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

Substantial variability exists in collateral density and ischemia-induced collateral growth among species. To begin to probe the underlying mechanisms, which are unknown, we characterized two mouse strains with marked differences in both parameters. Immediately after femoral artery ligation, collateral and foot perfusion were lower in BALB/c than C57BL/6 ( P < 0.05 here and below), suggesting fewer pre-existing collaterals. This was confirmed with angiography and immunohistochemistry (∼35% fewer collaterals in the BALB/c's thigh). Recovery of hindlimb perfusion was attenuated in BALB/c, in association with 54% less collateral remodeling, reduced angiogenesis, greater ischemia, and more impaired hindlimb use. Densities of CD45+and CD4+leukocytes around collaterals increased similarly, but TNF-α expression was 50% lower in BALB/c, which may contribute to reduced collateral remodeling. In normal tissues, compared with C57BL/6, BALB/c exhibit an altered arterial branching pattern and, like skeletal muscle above, have 30% fewer collaterals in intestine and, remarkably, almost none in pial circulation, resulting in greatly impaired perfusion after cerebral artery occlusion. Ischemic induction of VEGF-A was attenuated in BALB/c. Analysis of a C57BL/6 × BALB/c recombinant inbred strain dataset identified a quantitative trait locus for VEGF-A mRNA abundance at or near the Vegfa locus that associates with lower expression in BALB/c. This suggests a cis-acting polymorphism in the Vegfa gene in BALB/c could contribute to reduced VEGF-A expression and, in turn, the above deficiencies in this strain. These findings suggest these strains offer a model to investigate genetic determinants of collateral formation and growth in ischemia.

The generic limits of Hymenocallis have been variously proposed by different taxonomic workers, often without discussion or data. The genera Leptochiton, Ismene, Elisena, and Pseudostenomesson have been included with Hymenocallis, lumped together as the genus Ismene, or maintained as distinct genera. Recent cladistic analysis of plastid and nrDNA for Amaryllidaceae support a distinct tribe Hymenocallideae. Cladistic analyses of morphology, and plastid (trnL-F region) and nuclear ribosomal DNA (ITS) are presented alone and in combination for the tribe. Leptochiton is sister to the rest of the genera in the tribe in all analyses. While Hymenocallis is always resolved as monophyletic, Ismene is variably paraphyletic or monophyletic. The combined sequence data produce the most resolved and best-supported phylogeny, wherein Hymenocallis and Ismene are monophyletic sister genera. These data support an origin for the tribe in the Andes, with vicariant distribution of the largely Mesoamerican Hymenocallis. Formal recognition of Ismene subg. Elisena and Pseudostenomesson is established.