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Single ventricle (SV) congenital heart disease patients often form aortopulmonary collateral (APC) vessels via an unclear mechanism. To gain insights into APC pathogenesis, we correlated angiogenic factor levels with in vitro activity and angiographic APC assessment and examined whether SV patients have increased angiogenic factors that can stimulate endothelial cell sprouting in vitro.
In single ventricle (n=27) and biventricular acyanotic control patients (n=21), hypoxia inducible angiogenic factor levels were measured in femoral venous and arterial plasma at cardiac catheterization. To assess plasma angiogenic activity, we utilized a 3-D in vitro cell sprouting assay that recapitulates angiogenic sprouting. APC angiograms were graded using a 4-point scale.
Compared to controls, SV patients had increased vascular endothelial growth factor (VEGF) (artery: 58.7 ± 1.2 vs. 35.3 ± 1.1 pg/mL, p<0.01; vein: 34.8 ± 1.1 vs. 21 ± 1.2, p<0.03), stromal derived factor (SDF-1a) (artery: 1901.6 ± 1.1 vs. 1542.6 ± 1.1 pg/mL, p<0.03; vein: 2092.8 ± 1.1 vs. 1752.9 ± 1.1, p<0.02), and increased arterial soluble fms-like tyrosine kinase-1 (sFlt-1), a regulatory VEGF receptor (612.3 ± 1.2 vs. 243.1 ± 1.2 pg/mL, p<0.003). Plasma factors and sprout formation correlated poorly with APC severity.
We are the first to correlate plasma angiogenic factor levels with angiography and in vitro angiogenic activity in SV patients with APCs. SV patients have increased SDF-1a and sFlt-1 and their roles in APC formation require further investigation. Plasma factors and angiogenic activity correlate poorly with APC severity in SV patients suggesting complex mechanisms of angiogenesis.
Single ventricle (SV) heart disease is a broad category encompassing heterogeneous anatomy with an incidence of 0.005–0.01% in the general population.(1, 2) As these patients undergo palliative two or three-staged surgery in early life, aortopulmonary collateral (APC) blood vessel connections are frequently identified during pre-operative cardiac catheterization with an estimated prevalence of 46–71% after stage two palliation and 20–30% after stage three.(3–6)
Our current understanding of how and why APCs form is limited by conflicting data. APCs are suspected to form in part due to hypoxia-inducible angiogenic factors such as vascular endothelial growth factor (VEGF).(7–9) Prior studies documented elevated VEGF levels in children with cyanotic heart disease and SV patients with APCs.(8, 10) A separate study found that serum from cyanotic patients could promote primitive endothelial tube formation in vitro via VEGF signaling.(9) However, other studies found no relation between VEGF and the presence and/or severity of APCs.(11, 12) Given our incomplete understanding of why APCs form, we currently rely on palliative therapies such as surgical ligation or catheter-based embolization with metal coils or polyvinyl alcohol particles. However, these are downstream solutions that do not target the primary pathogenic pathway and do not reliably reduce systemic to pulmonary shunting.(3)
Traditionally, APCs have been identified via invasive angiography, which requires non-trivial amounts of radiation. Since collateral burden is difficult to quantify by angiography, qualitative subjective grading systems are primarily used.(3, 13, 14) Phase-contrast magnetic resonance imaging (MRI) is a noninvasive radiation-free method that can quantify systemic to pulmonary collateral blood flow.(15, 16) However, MRI often requires sedation and is limited by previously implanted hardware (e.g. coils) that either introduces imaging artifact or is not MRI-safe (15); although platinum devices can be used to avoid artifact and stainless steel devices (typically coils) are MRI-safe once endothelialized.
In this study, we hypothesized that functional angiogenic activity of plasma from single ventricle patients with APCs (SV-APC) would correlate with disease severity. This would make it feasible to develop a blood-based angiogenesis assay to quantify collateral burden without radiation or sedation. More importantly we might gain mechanistic insights into how APCs form to develop more effective therapies. As endothelial sprouting is an initial step in forming an immature capillary network, we suspected that APCs may form through a similar pathological mechanism, and we sought to determine whether plasma in SV patients with APCs can stimulate cell sprouting in vitro. To this end, we used a modified 3-dimensional in vitro endothelial cell sprouting assay that recapitulates angiogenic sprouting. We used this assay to characterize the angiogenic activity of SV patients with APCs and hypothesized that patients with greater APC severity would exhibit greater cell sprouting in vitro.
To identify potential causal factors for diagnosis and treatment of APCs, we also assayed the following factors that are known experimentally and clinically to be involved in sprouting angiogenesis to correlate with our cell sprouting data: VEGF (isoforms A, C, and D), stromal derived factor (SDF-1a, also known as CXCL12), fibroblastic growth factor (bFGF), and soluble fms-like tyrosine kinase (sFlt-1 or VEGF receptor 1).(8, 9, 17–24)
All patients undergoing aortography during elective cardiac catheterization at Children’s National Health System were eligible for enrollment. All patients were approached for enrollment just prior to their procedure and provided consent for intra-procedural blood collection. We excluded patients with conditions that could confound plasma factor level measurements such as disorders of hemostasis, malignancy, protein-losing enteropathy, and active infection or inflammation (e.g. myocarditis or Kawasaki’s disease). We also excluded patients with central lines as they require heparin which affects angiogenic factor levels.(10) Finally, we excluded patients with tetralogy of Fallot, pulmonary atresia and major aortopulmonary collateral arteries, because these collateral arteries (present in utero) may reflect a different disease process.(25) Patients were enrolled from March 2014 until March 2015. This study was approved by the institutional review board of Children’s National Health System (Washington, D.C.) and the Institutional Biosafety Committee of the National Heart, Lung and Blood Institute of the National Institutes of Health (Bethesda, MD).
Two blinded interventionists (JPK, KR) independently graded all aortic angiograms using the 4-point scale described by Spicer et al (Figure 4A).(13) Grade 1 and 2 collateral vessels did not opacify pulmonary arteries or veins, while grade 3 and 4 vessels caused pulmonary vasculature opacification. Grade 1 described a few (i.e. ≤ 3) collateral vessels that were small (< 1 mm). Both grade 2 and grade 3 described multiple (i.e. > 3) collateral vessels that were small or few vessels that were large. Grade 4 severity described multiple large vessels. Inter-rater reliability was calculated and scores were averaged when there was disagreement. Following study enrollment and blood collection, three patients (two SV-APC patients and one control) had left ventricular angiograms instead of aortic angiograms.
Blood was drawn from the femoral artery and vein following placement of access sheaths and prior to heparin administration due to its effect on factor levels.(10) A small amount of venous blood was used for complete blood count with differential analysis. The remaining venous and arterial blood was aliquoted into K3 EDTA vacutainers for plasma isolation (BD, Franklin Lakes, NJ). All blood was centrifuged within one hour of collection at 1100–1300 g for about 15 minutes. Additional centrifugation at 10000 g for 10 min was performed for all SDF-1a plasma to remove platelet fragments per manufacturer instructions. All isolated plasma was stored immediately at −80°C for later use in enzyme-linked immunosorbent assays (ELISA) and cell sprouting experiments. Total protein analysis was performed on leftover plasma from ELISA and sprouting experiments to ensure similar levels between both patient groups.
Colorimetric ELISA kits were used for SDF-1a (R&D Systems, Minneapolis, MN) and multi-analyte electrochemoluminescence ELISA kits were used for VEGF-A, C, & D, sFlt-1, and bFGF (MesoScale Discovery, Rockville, MD). All assays were run according to the manufacturer instructions. All plasma samples were assayed in duplicate and averaged. The lower limits of quantification (in pg/mL) were 47 for SDF-1a, 5 for VEGF-A, 146 for VEGF-C, 67 for VEGF-D, 10 for sFlt-1, and 3 for bFGF.
We modified a previously reported 3D sprouting assay to develop an assay utilizing human umbilical vein endothelial cells (HUVEC) in a collagen matrix.(26) Complete details are available in the Appendix. In summary, commercially available pooled donor HUVECs were cultured into macrospheres of ≈2000 cells, placed into a 3D collagen matrix, and incubated in the presence of 1 uL of venous or arterial patient plasma diluted in 2 mL of serum-free and factor-free endothelial cell basal media at 37ºC for 20–21 hours. Spheres were fixed with 4% PFA, stained with fluorescent dyes (Topro3, Phalloidin) and visualized using a confocal microscope. The number of induced sprouts was counted manually. Each sample was tested in triplicate and averaged.
All continuous data (e.g. number of sprouts, biomarker [factor] levels) are summarized as mean values with 95% confidence intervals and were analyzed using unpaired Students t-test after log-transformation to meet the normality assumption. Age was analyzed using Wilcoxon’s rank sum test and all values represent age-adjusted estimates with their corresponding p-values. The magnitude and statistical significance of association were evaluated using repeated measures linear regression for continuous parametric data and Spearman’s rank correlation analysis for non-parametric data. Inter-rater reliability in scoring angiograms was calculated using a kappa statistic. Using the significance level of 0.05, the study sample size provided 80% power to detect an effect size difference of 0.7 standard deviation (SD) for number of sprouts formed and 0.8 SD for ELISA measurements. All data were analyzed using Stata 13 software (StataCorp, College Station, TX).
Fifty-four consecutively consenting patients were enrolled, and six patients were excluded: two patients did not undergo aortograms following blood collection, two SV-APC patients were acyanotic, one SV-APC patient underwent orthotopic heart transplantation less than one year before enrollment, and one patient had abnormally low total protein. Of the remaining patients, 21 were acyanotic controls with biventricular circulation and 27 had single ventricle physiology. Summary and individual demographic data are provided in Tables 1 & 1S. Miscellaneous control group diagnoses included mitral stenosis/regurgitation, d-transposition of the great arteries status post arterial switch operation, repaired truncus arteriosus, and aortic atresia status post Yasui and Rastelli procedures. Miscellaneous SV group diagnoses included atrioventricular discordance with L-looped ventricles and pulmonary atresia, and heterotaxy with unbalanced atrioventricular canal.
Among the 48 graded angiograms, disagreement occurred in 4 SV-APC patients and 2 controls (12.5 %) producing a high inter-rater reliability (unweighted κ =0.81, weighted κ =0.89; Table 1S). In four cases (3 SV-APC, 1 control), the Spicer scores differed by 1, and in two cases (1 SV-APC, 1 control), the scores differed by 2. In the SV-APC group, 18 of 27 (66.7%) were rated as grade 3, two were grade 4, two were grade 2, one was grade 1 and one had no observable collaterals. Due to averaging, three patients were classified as grades 0.5, 2.5 and 3.5. Among controls, three patients had collaterals with average grades of 1, 1.5, and 2 (Table 1S). The presence of a PDA (n=3) or a mBTS (n=1) in some cases made it difficult to ascertain for pulmonary vessel opacification and assign a Spicer score. However, no collateral vessels were identified in the PDA patients and grade 1 collaterals were identified by one reviewer in the mBTS patient.
We measured arterial and venous plasma levels of the following hypoxia-inducible factors, which are known to be involved in sprouting angiogenesis: VEGF-A, C, D, bFGF, SDF-1a, and sFlt-1 (Figure 1).(17) Compared to controls, SV-APC patients had significantly higher arterial VEGF (a.k.a. VEGF-A) (58.7 pg/mL, 44 to 78.2 vs. 35.3 pg/mL, 29.3 to 42.6; p<0.01) and venous VEGF (34.8 pg/mL, 26.3 to 46 vs. 21 pg/mL, 15.6 to 28.2; p<0.03). SV-APC patients also had significantly increased arterial SDF-1a (1901.6 pg/mL, 1694.5 to 2134 vs. 1542.6 pg/mL, 1355 to 1756.2; p<0.03) and venous SDF-1a (2092.8 pg/mL, 1880.9 to 2328.6 vs. 1752.9 pg/mL, 1584.9 to 1938.8; p<0.02). Finally SV-APC patients had increased arterial sFlt-1 (612.3 pg/mL, 399 to 939.6 vs. 243.1 pg/mL, 173.7 to 340.1; p<0.003). Although most VEGF-C measurements fell below the assay’s lower limit of quantification (146 pg/mL), SV-APC patients had significantly increased arterial (84.6 pg/mL, 67.4 to 106.1 vs. 44.3 pg/mL, 33.8 to 58.1; p<0.001) and venous (81.2 pg/mL, 65.2 to 101.1 vs. 47.5 pg/mL, 38.4 to 58.9; p<0.002) levels. No other factors were significantly different between groups. In all instances, there was wide variability in SV-APC patient factor levels and considerable overlap with controls. Total protein levels were similar in both groups (SV-APC: 5.3 g/dL, 5.2 to 5.5; control: 5.3 g/dL, 5.1 to 5.4, p=0.49). Although hemoglobin and hematocrit were significantly elevated in the SV-APC group (data not shown, p<0.001), leukocytes (p=0.44) and platelet (p=0.58) counts were not different.
We evaluated the effects of arterial and venous plasma on inducing HUVEC sprout formation in our 3D culture system (Figure 2). Initially, we validated our assay by demonstrating robust cell sprouting in the presence of endothelial cell growth media which contains several angiogenic factors (e.g. VEGF and bFGF) and little to no sprouting with factor-free and serum-free basal media (Figure 2E and E′). We also found that plasma from a healthy volunteer can stimulate sprout formation in a dose dependent manner (data not shown).
While a few SV-APC patients exhibited high sprouting activity (Figure 2A, 2A′, 2B and 2B′) compared to control patients (Figure 2D and 2D′), other SV-APC patients did not (Figure 2C and 2C′). There was a wide variability in sprouting activity in the SV-APC group, which overlapped considerably with control patients. While there was a trend towards increased mean number of sprouts formed in the SV-APC group, this was not statistically significant in both artery (p=0.082) and vein (p=0.073) (Figure 2F).
Due to their involvement in other pathologic conditions, we explored whether plasma VEGF, SDF-1a and sFlt-1 levels were associated with cell sprouting activity in vitro and/or clinical APC severity as assessed by the Spicer score. In the entire study cohort, venous cell sprouting was significantly positively correlated with VEGF (p<0.01) (Figure 3). A similar positive correlation was observed with venous SDF-1a, which was not statistically significant (p=0.06). No associations were found with arterial data. Regardless of sampling site, APC severity scores did not correlate with cell sprouting activity and factor levels (Figure 4B–E).
To our knowledge, this is the first study to correlate angiographic assessment of APC burden with plasma hypoxia inducible factor measurements and in vitro angiogenic activity of SV patient plasma. We hypothesized that these patients would have significantly increased plasma levels of several hypoxia inducible factors, which would stimulate increased cell sprouting in vitro. Surprisingly, we found a wide range of in vitro angiogenic activity and factor levels that overlap considerably with control patients and correlate poorly with APC severity suggesting a complex mechanism of angiogenesis (Figures 1, ,22 & 4). We have demonstrated for the first time that SV patients have significantly elevated arterial and venous SDF-1a, and arterial sFlt-1, both of which may participate in APC formation.
We considered confounding variables that could have impacted our results. For instance, age was significantly different between SV-APC and control groups. A relationship between age and VEGF has been observed in early infancy; however, this does not appear to be an issue beyond age three months, which is when all study patients were sampled.(8, 12) We also adjusted for age in all of our analyses, to minimize its impact as a confounding variable. Additionally, the range of VEGF levels we measured was somewhat lower than the range reported in most previous studies.(8, 10, 27) Whereas other studies analyzed serum, we analyzed plasma where VEGF values are often lower; plasma measurements are considered more physiologically representative than serum, and our results resemble those of a previous study that also analyzed plasma.(12, 28) We also showed that total protein in both patient groups are similar and are likely not confounding factor level measurements. Finally, it is also possible that our HUVEC assay may not adequately recapitulate APC pathogenesis, and alternative mechanisms such as enlargement of existing vascular connections (e.g. bronchial arteries) may exist. However, the positive correlation we observed with plasma VEGF and cell sprouting supports the validity of our assay in assessing plasma angiogenic activity.
It is possible that factors other than the ones we studied are involved in APC pathogenesis. If the right factors were selected however, a difference between SV-APC and control patients could still have been masked given the time course during which angiogenesis occurs. Specifically, our data suggest a mechanism where upregulation and elevation of plasma angiogenic factors to create APCs may be a transient rather than a persistent state (Figure 4F). Physiologically, the time at which patients were enrolled and sampled is arbitrary and may have occurred during an angiogenically active or dormant phase. The significant relationship we observed between cell sprouting and VEGF suggest that we sampled patients from both phases (Figure 3). In a dormant state, previously formed collaterals may be visualized angiographically while plasma angiogenic activity (e.g. factor levels and sprouts formed) is low. Similarly, in an angiogenically active state, there may be a time lag from when angiogenic activity peaks to when collateral vessels are visible angiographically. This is supported by the poor correlation of APC Spicer score with cell sprouting and plasma factor levels (Figure 4B–E).
Although all the factors we assayed are regulated by the transcription factor hypoxia inducible factor 1-alpha (HIF-1a), a relationship between systemic hypoxemia and factor levels has been inconsistently documented.(11, 12, 27) Our proposed mechanism where factors downregulate back to normal levels, due to a stimulus like chronic hypoxia could explain these conflicting results. This phenomenon has been previously demonstrated in patients with chronic limb ischemia.(24) Our hypothetical mechanism could also explain why some investigators found a relationship between VEGF and APCs while others did not.(10–12) Although we have graphically depicted only one peak (Figure 4E), the true pathophysiology may better resemble a sine wave with multiple peaks and valleys. Conflicting observations regarding the relationship of patient age (i.e. duration of hypoxemia) to APC severity support a model where angiogenic processes may get turned on and off over time.(29, 30) If APC pathogenesis is cyclical as we propose, this has important implications for diagnosis and treatment of patients who have previously formed APCs but are now angiogenically dormant. Blood based angiogenesis assays or biomarker tests could have poor sensitivity in these patients limiting their utility. Similarly, although anti-angiogenic agents such as bevacizumab could have therapeutic potential by blocking VEGF signaling, they may not affect APCs that were previously formed.
The source of plasma angiogenic factors that form APCs is still unclear. Prior studies found no significant differences in respective factor levels when sampling blood in APC patients from the superior and inferior vena cavae as well as from a systemic artery. (8, 10) In the present study, we were surprised to find such variation in factors such as VEGF and sFlt-1 levels between the femoral artery and vein in both patient groups. Although identifying the mechanism for this variation is outside the scope of our present study, the trends we observed may help localize a source of these factors. From a practical standpoint, knowing the source will determine whether a reliable biomarker can be identified from venous or arterial blood.
Angiogenesis is a complex process requiring many steps and factors. Among the angiogenic factors previously studied in APC patients, only VEGF has emerged as a candidate pathogenic factor in some but not all studies. To our knowledge we are the first to report significantly increased levels of arterial and venous VEGF together with arterial sFlt-1 (also known as VEGF receptor 1) as well as increased arterial and venous SDF-1a. VEGF primarily signals by binding to VEGF receptor 2 (VEGFR2), while the role of sFlt-1 is less understood. Flt-1 exists in both membrane bound and soluble forms (sFlt-1) and is traditionally thought to exert anti-angiogenic effects by trapping and preventing circulating VEGF from signaling via VEGFR2.(17) However, several studies support a potential pro-angiogenic role for sFlt-1. As endothelial cells sprout from a parent vessel to create branches, guidance cues are necessary to direct elongation so emerging sprouts will not curl back on themselves or collide with other sprouts.(17) In embryonic murine stem cells sFlt-1 knockout caused endothelial sprouts to emanate randomly without a direction; transgenic endogenous sFlt-1 expression rescued this phenotype by restoring a direction for elongation.(20) Soluble Flt-1 has also been shown to promote interactions between endothelial and mural cells leading to vessel maturation and stability.(22) Furthermore, elevated sFlt-1 levels have been measured in patients with other hypoxic and inflammatory diseases such as pre-eclampsia and sepsis.(21, 23) Hence, although sFlt-1 may not itself stimulate sprout formation, our results coupled with previous findings suggest that it may help provide order, directionality, and stability during APC sprouting angiogenesis. SDF-1a (a.k.a. CXC12) is a well-known hypoxia-inducible chemoattractant molecule whose role in vascular formation has been well documented in the gut, kidney, and retina.(31–33) Its pro-angiogenic effect involves recruiting and retaining pro-angiogenic bone marrow derived cells to areas of active angiogenesis.(17) Using homozygous knockout of SDF-1a or its receptor CXCR4, we previously demonstrated that this signaling axis works together with VEGF to promote nerve-vessel alignment as well as arteriogenesis in murine embryonic limb skin.(18) Increased expression of VEGF and SDF-1a has also been demonstrated in ischemic versus nonischemic muscle in patients undergoing limb amputation for peripheral vascular disease.(24) Finally, increased VEGF, SDF-1a, and circulating endothelial progenitor cells have been documented in cyanotic children with tetralogy of Fallot.(19) These results together with our findings suggest that sFlt-1 and SDF-1a may be involved in APC formation.
Our current understanding of aortopulmonary collateral vessel pathogenesis is limited. The present study has demonstrated that single ventricle patients exhibit a wide range of plasma angiogenic activity that correlates poorly with disease severity, which may be explained by a time dependent phenomenon. If true, this would limit the utility of blood-based methods of diagnosing APCs. Stromal derived factor and soluble flt-1, a regulatory VEGF receptor, are elevated in SV patients and their roles in APC pathogenesis require further investigation.
A major limitation of our study is that invasive contrast angiography can have poor sensitivity in detecting APC severity due to variations in technique.(3, 5, 6, 13) The Spicer scoring system, although practical, is an ordinal and subjective scale which does not represent the full spectrum of disease.(13) It also does not assess for other pathologic angiogenesis unrelated to APCs such as pulmonary arteriovenous malformations (seen in single ventricle patients) or physiologic angiogenesis in controls. The presence of pulmonary arteriovenous malformations could have contributed to the poor correlation of APC Spicer score with cell sprouting and plasma factor levels. Although all patients would have ideally undergone phase-contrast MRI to quantify APC flow, this is not a routine clinical practice at our institution. A second limitation arises from inherent variability between groups with respect to age as well as within groups in terms of diagnosis and anatomy, which increases susceptibility to confounding variables. These various heterogeneities may weaken the strength of the various differences and correlations (or lack thereof) we reported. These limitations arise in part from our inability to perform invasive blood sampling and aortography in age-matched healthy volunteers. Further, all study patients would have ideally been sampled at multiple time points, which is not ethically and practically feasible. Without this data, we cannot definitively support our proposed time course mechanism of APC formation. Finally, despite our efforts to standardize all cell sprouting experiments, we acknowledge that slight differences in conditions from run to run could have potentially confounded our sprouting data. Since we are the first to use the cell sprouting assay with human plasma the expected magnitude of difference in sprouting activity is not known and our study may have been underpowered to detect a difference.
Nefthi Sandeep was supported by an institutional Ruth L. Kirschstein National Research Service Award (T32) and National Center for Advancing Translational Sciences (UL1TR000075). This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health (HL006116-05 to Y.-s.M.).
We thank the many individuals that greatly supported and/or contributed to this project: CNHS Division of Hematology: Naomi Luban and Lori Luchtman-Jones provided project funding and mentorship. CNHS Division of Cardiology: Catherine Connors, Ileen Cronin, Charles Fleming, Manuel Mendez III, and Beth Thompson performed blood collections. Lowell Frank and E. Anne Greene provided scholarly guidance. CNHS Clinical Research Center: Pablo Cure and Amanda Kasper provided additional sources of intramural funding. Jayna Bryant, Vonterris Hagan-Temple, Ianka Laidlow, Marlene Lee, Brenda Martin, Vera Okoye, and Lisa Pickett-Evans performed blood processing and plasma isolation. National Heart, Lung, and Blood Institute: Faye Baldrey and Rachel Reed provided administrative assistance. Christian Combs, Julia Doveikis, Krista Gill, Christopher Hourigan, Nehal Mehta, Balaji Natarajan, Martin Playford, Shawn Rose and Mikhael Wallowitz (Mesoscale Discovery) provided technical expertise, equipment, and consultations. We also thank the other members of Laboratory of Stem Cell and Neuro-Vascular Biology for technical help and thoughtful discussion. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health as well as Award Number UL1TR000075 from the NIH National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.
To standardize all experiments as much as possible all HUVECs were derived from a commercially available single vial of pooled donor primary cells (Lonza Ltd, Basel, Switzerland). Cell colonies were expanded and grown from this vial and passaged three times over nine days in 55 cm2 culture dishes (Corning Life Sciences, Tewksbury, MA) before being stocked into liquid nitrogen. Growth never exceeded 70–80% confluency. For each experiment, cells were plated onto 55 cm2 culture dishes with endothelial cell growth media (Lonza Ltd, Basel, Switzerland). After three days, cells were washed, trypsinized, counted, resuspended in endothelial growth media, and cultured in a 96-well clear round bottom ultra-low attachment microplate (Corning Life Sciences, Tewksbury, MA) for 27–28 hours to produce macrospheres comprising ≈2000 cells. Macrospheres were gently embedded into a 3D collagen matrix (Nitta Gelatin, Osaka, Japan) and applied to 9.6 cm2 glass bottom culture dishes (In Vitro Scientific, Sunnyvale, CA). After incubation at 37°C with 5% CO2 for 30–40 minutes, 1 uL of patient plasma diluted in 2 mL of serum-free and factor-free endothelial cell basal media (Lonza Ltd, Basel, Switzerland) was applied to each collagen-embedded HUVEC macrosphere. All plasma samples were assayed in triplicate. After incubating for 20–21 hours at 37°C with 5% CO2, cells were fixed with 4% paraformaledyde, stained with fluorescent dyes (To-pro-3, pan-nuclear marker and Phalloidin, actin cytoskeleton marker), visualized using a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany), and the number of induced sprouts was manually counted and averaged. To minimize run to run variability, SV-APC and control patients were run in the same experiments as much as possible. HUVEC macrospheres were tested in every experimental run with angiogenically inert serum-free and factor-free basal media (negative control) and serum and growth factor containing endothelial cell growth media (positive control, Figure 2E and E′).
No potential conflicts of interest exist
None of the authors has any financial or other conflicts of interests.
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