DOI: 10.5061/dryad.b2rbnzsgw
title: Filgrastim, fibrinolysis, and neovascularization

description: Objective: Segmental recanalization of chronically occluded arteries was
 observed in patients with chronic limb-threatening ischemia (CLTI) treated
 with Filgrastim, a granulocyte colony stimulating factor, every 72 hours
 for up to a month, and an infra-geniculate programmed compression
 pump (PCP) for 3 hours daily. Molecular evidence for fibrinolysis and
 neovascularization was sought. Methods: CLTI patients were treated with
 PCP alone (N=19), or with Filgrastim and PCP (N=8 and N=6, at 2
 institutions). ELISA was used to measure the plasma concentration
 of Plasmin and of Fibrin Degradation Products (FDP), and the serum
 concentration of proteins associated with neovascularization. In the PCP
 alone group blood was sampled on day 1 (baseline) and after 30 days of
 daily PCP. In the Filgrastim and PCP group blood was drawn on day 1, and
 one day after the 5th and the 10th Filgrastim doses. Each blood draw
 occurred before and after 2 hours of supervised PCP. Results: Significant
 (p&lt;0.01) PCP independent increases in the plasma concentration of
 plasmin (&gt;10 fold) and FDP (&gt;5 fold) were observed one day
 after both the 5th and the 10th Filgrastim doses, compared to day 1.
 Significant (p&lt;0.05) increases in the concentration of
 pro-angiogenic proteins (e.g., HGF, MMP-9, VEGF A) were also observed.
 Conclusion: Filgrastim at this novel dosimetry induced
 fibrinolysis without causing acute hemorrhage, in addition to
 inducing a pro-angiogenic milieu conducive to NV. Further clinical testing
 is warranted at this novel dosimetry in CLTI, as well as in other
 chronically ischemic tissue beds.
description: Population: CLTI
 patients who were not candidates for, or had failed, previous invasive
 revascularization procedures. Inclusion criteria:
 Patients complaining of ischemic forefoot rest pain, gangrene, and/or
 ischemic ulceration were enrolled if the ankle brachial arterial index was
 &lt;0.45. Exclusion criteria:
 Acute limb ischemia, non-salvageable extremity, untreated hypercoagulable
 disorder, sickle cell disease, myeloproliferative disorder, dialysis,
 creatinine above 3.5 mg/dl, active cancer, dementia, non-compliance,
 intolerance of PCP, body mass index over 34, venous stasis ulcer, history
 of lymphoma or leukemia, uncorrected symptomatic coronary artery disease,
 severe carotid stenosis, sepsis proximal to the forefoot, allergy to
 Filgrastim. Patient groups:
 Table 2 lists the patient characteristics. Blood assays were performed on
 2 patient groups. The first group was treated at a university-affiliated
 institution (UC, 2012-13) with PCP alone (N=19). The second group (N=14)
 was treated with PCP and Filgrastim: six at another independent
 university-affiliated institution (UIC, 2016-19) and eight at a hospital
 (2014-15). Filgrastim: Each
 patient was to receive Filgrastim subcutaneous injections, one every 72
 hours at approximately 10 mcg/kg for up to ten doses. Filgrastim
 (Neupogen) was purchased from Amgen Inc (Thousand Oaks, CA) in 480 and 300
 mcg vials. These were kept refrigerated between 2°C and 8°C and stored in
 the dark. Small losses of agent occurred during administration, which
 typically required two separate injections in the subcutaneous tissue in
 the lower abdomen. Fourteen patients received 5 doses. Ten of the 14
 received all 10 doses. Mobilized progenitor cells circulated until either
 being engrafted or otherwise cleared from the circulation. The leukocyte
 count returned to normal approximately every 72 hours. The product label
 for Filgrastim lists progenitor cell mobilization as an indication (not
 CLTI). The FDA granted a waiver to use Filgrastim at the novel dosimetry
 in CLTI patients at both institutions. Filgrastim has been safely used in
 healthy patients for the purpose of bone marrow harvest of mobilized
 progenitor cells.[i] 
 Oncologists have nearly 3 decades of experience with
 Filgrastim. Initial
 concerns over promoting tumor angiogenesis were not substantiated [ii].
 While severe complications are uncommon, they can be
 significant (e.g., capillary leak syndrome, myocardial infarction, and
 splenic rupture). Hemorrhage is not a reported side effect; fibrinolysis
 and NV are not listed on the Filgrastim label. PCP: The ArtAssist
 device (ACI Medical LLC, San Marcos CA) applies sequential pressure to the
 calf, and foot. PCP was used at home on both legs in the seated position
 for 3 hours daily. Rapid inflation of pneumatic cuffs (0 to 120 mmHg in
 &lt;0.3 sec) provides an endothelial shear stress stimulus, while at
 the same time driving in oxygenated nutritive blood flow and facilitating
 venous return (Table 1). The pressure is held in each of the cuffs for
 three seconds. Rapid deflation follows. Three cycles occur per
 minute. Phlebotomy: Group 1
 patients were treated with PCP alone, and had blood drawn on Day 1 and Day
 30. Group 2 patients were treated with Filgrastim and PCP and had three
 blood samples drawn: on Day 1, on the day after the
 5<sup>th</sup> Filgrastim dose, and on the day after the
 10<sup>th</sup> Filgrastim dose. In both Groups, on each
 day, blood was drawn using a 21- gauge butterfly inserted before and again
 after 2 hours of observed PCP. Blood was collected into serum separator
 and EDTA plasma separation tubes. Specimens were immediately transported
 on ice to the independent laboratories at the university-affiliated
 institutions. Serum
 separation: Whole blood was collected in serum separator tubes.
 Following clot formation (30-60 min) each tube was centrifuged for 15 min
 at 1000g. Serum aliquots were stored at -80°C in labeled cryovials (to
 avoid repetitive freeze/thaw) until batch analysis. Plasma separation
 was used for preparation of samples for ELISA, and for cytometry. Whole
 blood was collected in EDTA-treated tubes. Red cells and
 platelets were removed after centrifugation at 2,000g for 20 minutes at
 4<sup>o</sup>C. Plasma aliquots were stored at
 -80°C. Enzyme-linked
 immunosorbent assay (ELISA): Protein concentrations were measured in
 duplicate in plasma or in serum using Human ELISA Kits according to each
 kit’s instructions (Table 7). Plasmin and Fibrin Degradation Products
 (FDP) were measured in
 plasma at UIC and in serum at UC to assess fibrinolysis.
 Proteins associated with NV included HGF[iii], VEGF-A[iv],  MMP-9[v], Angipoietin-1 [vi],PDGF-AA, PDGF-BB, PDGF-AB [vii],TNF-a[viii], MCP-1[ix] and TGF-b[x], PLGF[xi], IL-6[xii], and IGF-1 [xiii]. All patient-specific samples were analyzed together using a single ELISA kit. Cytometry: Mature endothelial CD31+ (PECAM-1), progenitor CD34+, and endothelial progenitor CD309+ (VEGFR2+) cell populations were measured (Table 7). White Blood Cell (WBC) and differential counts were obtained on plasma in the hospital hematology laboratories. Serum nitrite: Serum nitrite, a breakdown product of nitric oxide (NO), reflects nitric oxide synthase (NOS) activity. It was measured before and after 2 hours of supervised PCP with a quantitative fluorometric assay based on the reaction of nitrite with 2,3- diaminonaphthalene under acidic conditions to form fluorescent 1-(<em>H</em>)-naphthotriazole (Table 7). Statistics: Each patient had 2 internal controls. The first internal control was obtained on Day 1, prior to PCP or Filgrastim. Changes resulting from daily PCP use for 30 days (Group 1) or caused by Filgrastim (Group 2) were referenced to data from Day 1. The second control was obtained on arrival (at T=0) to each 2 hour observed PCP session, and was used to ascertain the effect of PCP (at T=2HR) on Day 1 for all patients, on Day 30 for the PCP alone group, and on the day after the 5<sup>th</sup> and the 10<sup>th</sup> doses of Filgrastim in the Filgrastim+ PCP group.  Percent changes are reported relative to each of these two controls (change from control divided by the control). The paired Student t-test was used to estimate a P value directly from the ELISA data (not from the percent changes). An unpaired T-Test was used when comparing the aggregated ELISA data in all patients obtained before any Filgrastim exposure to all data obtained after Filgrastim was given. P values &lt;0.05 are considered significant. The significant findings in the UC study led to the confirmatory laboratory study at UIC in 6 CLTI patients. The UIC sample size was calculated from the UC data to provide 80% power to detect changes in protein levels of 0.66 standard deviations assuming a two-sided significance level of 0.05. [i] BE Shaw, DL Confer , W Hwang, MA Pulsipher. A review of the genetic and long-term effects of G-CSF injections in healthy donors: a reassuring lack of evidence for the development of haematological malignancies. Bone Marrow Transplantation (2015) 50, 334–340; doi:10.1038/bmt.2014.278; [ii] Okazaki T, Ebihara S, Asada M, Kanda M, Sasaki H, Yamaya M. Granulocyte colony-stimulating factor promotes tumor angiogenesis via increasing circulating endothelial progenitor cells and Gr11CD11b1 cells in cancer animal models. International Immunology, Vol. 18, No. 1, pp. 1–9. doi:10.1093/intimm/dxh334 [iii] Kaga T, Kawano H, Sakaguchi M, Nakazawa T, Taniyama Y, Morishita R. Hepatocyte growth factor stimulated angiogenesis without inflammation: differential actions between hepatocyte growth factor, vascular endothelial growth factor and basic fibroblast growth factor. Vascul Pharmacol. 2012 Aug 19;57(1):3-9. doi: 10.1016/j.vph.2012.02.002. Epub 2012 Feb 14. PMID: 22361334. [iv] Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Manseau EJ, Dvorak AM, Dvorak HF. VEGF-A induces angiogenesis, arteriogenesis, lymphangiogenesis, and vascular malformations. Cold Spring Harb Symp Quant Biol. 2002;67:227-37. doi: 10.1101/sqb.2002.67.227. PMID: 12858545. [v], Wang X, Khalil RA. Matrix Metalloproteinases, Vascular Remodeling, and Vascular Disease. <em>Adv Pharmacol</em>. 2018;81:241-330. doi:10.1016/bs.apha.2017.08.002 [vi] Koh GY. Orchestral actions of angiopoietin-1 in vascular regeneration. Trends Mol Med. 2013 Jan;19(1):31-9. doi: 10.1016/j.molmed.2012.10.010. Epub 2012 Nov 23. PMID: 23182855. [vii] Hellberg C, Ostman A, Heldin CH. PDGF and vessel maturation. Recent Results Cancer Res. 2010;180:103-14. doi: 10.1007/978-3-540-78281-0_7. PMID: 20033380. [viii] Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, Nuseir N. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha. Nature. 1987 Oct 15-21;329(6140):630-2. doi: 10.1038/329630a0. PMID: 2443857. [ix] Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4678-82. doi: 10.1073/pnas.91.11.4678. PMID: 8197118; PMCID: PMC43851. [x] Goumans MJ, Liu Z, ten Dijke P. TGF-beta signaling in vascular biology and dysfunction. Cell Res. 2009 Jan;19(1):116-27. doi: 10.1038/cr.2008.326. PMID: 19114994. [xi] De Falco, S. The discovery of placenta growth factor and its biological activity. Exp Mol Med 44, 1–9 (2012). https://doi.org/10.3858/emm.2012.44.1.025 [xii] Villar-Fincheira P, Sanhueza-Olivares F, Norambuena-Soto I, et al. Role of Interleukin-6 in Vascular Health and Disease. <em>Front Mol Biosci</em>. 2021;8:641734. Published 2021 Mar 16. doi:10.3389/fmolb.2021.641734 [xiii] Lin S, Zhang Q, Shao X, Zhang T, Xue C, Shi S, Zhao D, Lin Y. IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway. Cell Prolif. 2017 Dec;50(6):e12390. doi: 10.1111/cpr.12390. Epub 2017 Sep 27. PMID: 28960620; PMCID: PMC6529130.
description: <strong>Guide</strong> Brackets: “[protein]” mean “concentration of” that protein T=0: first blood draw on a particular day T=2HR: blood draw after 2 hours of PCP Patients 1201-1219: PCP alone at a university-affiliated institution: <strong>Table Gaps</strong>: two patients stopped PCP before 30 days. Patients 1401 to 1408: Filgrastim+PCP at a hospital, with all assays done at a university-affiliated institution. <strong>Table Gaps</strong>: two patients stopped Filgrastim at 5 doses (1404 due to transfer out of state, and 1408 due to resolution of rest pain), and one at 7 doses (1406 due to intolerance of the PCP); one did not store the filgrastim correctly (1401) and had to start over, and one stopped at 5 doses only to resume several months later (1407). Patients P003-P009: Filgrastim+PCP at a university-affiliated institution:  <strong>Table Gap</strong>: P009 had only one blood draw on last day (T=0): did not wear PCP on that day.
description: # Filgrastim, fibrinolysis, and neovascularization Dataset DOI:
 [10.5061/dryad.b2rbnzsgw](10.5061/dryad.b2rbnzsgw) ## Description of the
 data and file structure The key hematologic and ELISA data were obtained
 in patients treated with Filgrastim and the ArtAssist Device together, and
 were derived from 2 independent university-affiliated institutions from
 patients with Chronic Limb Threatening Ischemia (CLTI) recruited from the
 Vascular Surgery practices at each institution. The control group of
 hematologic and ELISA data was obtained from patients treated with the
 ArtAssist Device alone. The goal was to measure hematologic (CD34, VEGFR2)
 and ELISA evidence supporting clinical evidence of neovascularization
 (VEGF, HGF, MMP9, PDGF...) in patient serum and of fibrinolysis (plasmin,
 Fibrin Degradation Products) in patient plasma and serum. Also, in the
 control group, the goal was to measure evidence of endothelial cell
 activation by the ArtAssist device, including MCP-1 serum level (ELISA),
 upregulation of nitric oxide synthase (serum nitrite level), and CD31
 (Cytometry). Throughout the datasets, PCP refers to Programmed Compression
 Pump; the one used was the ArtAssist device ### Files and variables ####
 File: 2026_10_Dryad_Tables_from_TERM_publication_by_Eton.xlsx
 **Description:**  **Table 1**- Rationale for a Novel Neovascularization
 Strategy. Neovascularization is a natural response to arterial occlusive
 disease. CLTI arises when neovascularization fails. Our strategy (Column
 3) was developed to overcome the obstacles listed in this table. The goal
 of this strategy is to help restore this natural process. **Table 2**-
 Patient Demographics. Column 1 is a 62 patient cohort treated with PCP
 alone at UC and does not include the 14 patients treated with PCP and
 Filgrastim in this study. Columns 2 to 4 represent the patients in whom
 the assays were performed: PCP alone (N=19), PCP and Filgrastim (N=14).
 Numbers in parentheses are standard deviation from the mean. **Table 3**-
 Effect of Filgrastim (paired to control data). The relative percent change
 in concentration was calculated using data from Day 1 (before Filgrastim
 and PCP) as baseline for each patient. Paired T-Tests using the
 concentrations were used to derive the P Values. [Plasmin] and [FDP] were
 measured in plasma at UIC and in serum at UC. The other proteins were
 measured in serum. P values &lt;0.05 are in bold. Despite the magnitude of
 increase, some did not reach significance due to the wide standard
 deviation (SD) in this small population. **Table 4**- Effect of PCP alone.
 The relative percent change in concentration was calculated using data
 from Day 1, T=0 (before onset of PCP) as baseline for each patient. Paired
 T-Tests using the concentrations were used to derive the P Values. P
 values &lt;0.05 are in bold. **Table 5**- Filgrastim effect in CLTI
 (unpaired Group analysis). This is a summary comparison of ELISA
 concentrations aggregated into 2 groups. Group A: no Filgrastim exposure
 (includes all Day 1 data on all patients, and Day 30 Data in the PCP alone
 group). Group B: all data obtained one day after both the 5th and 10th
 Filgrastim doses. P Values are derived from unpaired T Test of ELISA data.
 N is the number of ELISA results. **Table 6**- Filgrastim effect on
 circulatory ells in CLTI patients treated with Filgrastim and PCP.
 Cytometry confirmed the significant percentage increase in CD34+
 progenitor and VEGFR2+ endothelial progenitor cells one day after each
 Filgrastim dose. The Differential blood cell count also shows the
 percentage increase in white blood cell count (WBC) and neutrophils the
 day after the 5th Filgrastim dose. **Table 7**- Assays and Equipment ####
 File: 2025_06_10_DRYAD_Eton_APPENDIX_TABLES.xlsx **Description:**  **Table
 A1**- ELISA results for Plasmin in plasma and in serum. P values (paired
 TTest) were derived from the ELISA concentrations. Filgrastim
 significantly elevated [Plasmin] one day after the 5th and 10th doses
 (referenced to Day 1) at T=0 and T=2 hours (P values highlighted). Two
 hours of PCP had no significant impact. P008, 1403, and 1406 had recent
 thromboses, with an active fibrinolytic cascade on Day 1. **Table A2**-
 ELISA results for **FDP** in plasma and in serum. P values (paired TTest)
 were derived from the ELISA concentrations. Filgrastim significantly
 elevated [FDP] one day after the 5th and 10th doses (referenced to Day 1)
 at T=0 and T=2 hours (P values highlighted). Two hours of PCP had no
 significant impact. P008, 1403, and 1406 had recent thromboses, with an
 active fibrinolytic cascade on Day 1. **Table A3**- ELISA results for
 serum Hepatocyte Growth Factor (**HGF**). P values (paired TTest) were
 derived from the ELISA concentrations. Filgrastim significantly elevated
 [HGF] one day after the 5th and 10th doses (referenced to Day 1) at T=0
 and T=2 hours (P values highlighted). Two hours of PCP had no significant
 impact. **Table A4**- ELISA results for serum [**MMP-9**], diluted
 100-fold. P values (paired TTest) were derived from the ELISA
 concentrations. Filgrastim significantly elevated [MMP-9] one day after
 the 5th and 10th doses (referenced to Day 1) at T=0 and T=2 hours (P
 values highlighted). Two hours of PCP had no significant impact. **Table
 A5**- ELISA results for serum **VEGF-A**. P values (paired TTest) were
 derived from the ELISA concentrations. Filgrastim significantly elevated
 [VEGF=A] one day after the 5th and 10th doses (referenced to Day 1) at T=0
 and T=2 hours (P values highlighted) in the UIC group. Two hours of PCP
 had no significant impact. Patient 1406 had the lowest Filgrastim dose
 (6.6 mcg/kg), and 1407 had significantly elevated [VEGF-A] on Day 1,
 dampening the percent change after Filgrastim. **Table A6**- ELISA results
 for serum **Angiopoietin-1**. P values (paired TTest) were derived from
 the ELISA concentrations. Filgrastim significantly elevated
 [Angiopoietin-1] by the 10th dose (referenced to Day 1) at T=0 and T=2
 hours (P values highlighted). The percent change from 2 hours of PCP was
 not significant on any of the days (P&gt;0.05). **Table A7**- ELISA
 results for serum **PDGF-AA**. P values (paired TTest) were derived from
 the ELISA concentrations. Filgrastim significantly elevated [PDGF-AA] by
 the 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values
 highlighted). The percent change from 2 hours of PCP was not significant
 on any of the days (P&gt;0.05). **Table A8**- ELISA results for serum
 **PDGF-BB**. P values (paired TTest) were derived from the ELISA
 concentrations. Filgrastim significantly elevated [PDGF-BB] by the 10th
 dose (referenced to Day 1) at T=0 and T=2 hours (P values highlighted).
 The percent change from 2 hours of PCP was not significant on any of the
 days (P&gt;0.05). **Table A9**- ELISA results for serum **PDGF-AB**. P
 values (paired TTest) were derived from the ELISA concentrations.
 [PDGF-AB] increased after the 10th Filgrastim dose (referenced to Day 1)
 at T=0 (p=0.08) and T=2 hours (p=0.058), but the increase did not reach
 the significance threshold, likely due to the large SD and small N. The
 trend, however, is evident. The percent change from 2 hours of PCP was not
 significant on any of the days (P&gt;0.05). **Table A10**- ELISA results
 for serum **TNF-alpha**. P values (paired TTest) were derived from the
 ELISA concentrations. Filgrastim significantly elevated [TNF-alpha] by the
 10th dose (referenced to Day 1) at T=0 and T=2 hours (P values
 highlighted). The percent change from 2 hours of PCP was not significant
 on any of the days (P&gt;0.05). **Table A11**- ELISA results for serum
 **TGF-beta**. P values (paired TTest) were derived from the ELISA
 concentrations. Filgrastim significantly elevated [TGF-beta] by the 10th
 dose (referenced to Day 1) at T=2 hours (P value highlighted). The percent
 change from 2 hours of PCP was not significant on any of the days
 (P&gt;0.05). **Table A12**- ELISA results for serum **IGF-1**. P values
 (paired TTest) were derived from the ELISA concentrations. Filgrastim
 significantly elevated [IGF-1] by the 10th dose (referenced to Day 1) at
 T=0 in the UC dataset only (P value highlighted). The percent change from
 2 hours of PCP was not significant on any of the days (P&gt;0.05). **Table
 A-13**- ELISA results for serum **PLGF**. P values (paired TTest) were
 derived from the ELISA concentrations. [PLGF] increased after the 10th
 Filgrastim dose (referenced to Day 1) at T=0 (p=0.1) and T=2 hours
 (p=0.5), but the increase did not reach the significance threshold. The
 percent change from 2 hours of PCP was not significant on any of the days
 (P&gt;0.05). **Table A-14**- ELISA results for serum **IL-6**. P values
 (paired TTest) were derived from the ELISA concentrations. [IL-6]
 increased after the 10th Filgrastim dose (referenced to Day 1) at T=0
 (p=0.1) and T=2 hours (p=0.5), but the increase did not reach the
 significance threshold. The percent change from 2 hours of PCP was not
 significant on any of the days (P&gt;0.05). **Table A-15**- ELISA results
 for **MCP-1**. P values (paired TTest) were derived from the ELISA
 concentrations. [MCP-1] decreased after the 10th Filgrastim dose
 (referenced to Day 1) at T=0 (p=0.1) and T=2 hours but the decrease did
 not reach the significance threshold. The percent change from 2 hours of
 PCP was not significant on any of the days (P&gt;0.05). **Table A-16**-
 PCP alone  for 2 hours on Day 1 led to a 19%+32% increase (p=0.006) in
 serum [MCP-1] measured by ELISA (pg/ml). Values are listed prior to PCP
 (T=0) and after 2 hours of supervised PCP (T=2HR). (SD=Standard deviation)
 **Table A-17**- Baseline cytometry on Day 1 for the 19 patients treated
 with pump alone**.** **Table A-18**- **CD31+**, **CD34+,** and **VEGFR2+**
 Cell Fractions (10,000 cells) in each patient treated with Filgrastim and
 PCP. The percentage increase due to PCP (T=2HR vs T=0 on each Day) and to
 Filgrastim (Referenced to Day 1 data) is tabulated. SD=Standard Deviation
 **Table A-19**- Nitrite (nM/ml) progressively increased during 30 days of
 PCP alone, 3 hours daily. On Days 1 and 30, nitrite increased after 2 hour
 PCP. This assay does not distinguish between NO generation in patients
 produced by inflammatory cells (iNOS) versus NO produced from the
 endothelium (eNOS). **Table A-20**- Nitrite (nM/ml) progressively
 increased in patients receiving Filgrastim over the 10 dose course, and
 after  2 hour PCP sessions. [Nitrite] for each patient are listed prior to
 PCP (T=0) and after 2 hours of supervised PCP (T=2HR). Note the
 significant P values. (SD=Standard deviation) **Table A-21**- Effect of
 PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on
 [Plasmin] **Table A-22**- Effect of PCP ALONE for 2 HR on Day 1 and 30,
 and for 30 days (3 hours daily) on [FDP] **Table A-23**- Effect of PCP
 ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on [HGF]
 **Table A-24**- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30
 days (3 hours daily) on [MMP-9] **Table A-25**- Effect of PCP ALONE for 2
 HR on Day 1 and 30, and for 30 days (3 hours daily) on [VEGF-A] **Table
 A-26**- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3
 hours daily) on [Angiopoietin-1] **Table A-27**- Effect of PCP ALONE for 2
 HR on Day 1 and 30, and for 30 days (3 hours daily) on [IGF-1] **Table
 A-28**- Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3
 hours daily) on [PDGF-AA] **Table A-29**- Effect of PCP ALONE for 2 HR on
 Day 1 and 30, and for 30 days (3 hours daily) on [PDGF-BB] **Table A-30**-
 Effect of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours
 daily) on [PDGF-AB] **Table A-31**- Effect of PCP ALONE for 2 HR on Day 1
 and 30, and for 30 days (3 hours daily) on [MCP-1] **Table A-32**- Effect
 of PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on
 [TGF-beta] **Table A-33**- Effect of PCP ALONE for 2 HR on Day 1 and 30,
 and for 30 days (3 hours daily) on [TNF-alpha] **Table A-34**- Effect of
 PCP ALONE for 2 HR on Day 1 and 30, and for 30 days (3 hours daily) on
 [PLGF] **Table A-35**- Effect of PCP ALONE for 2 HR on Day 1 and 30, and
 for 30 days (3 hours daily) on [IL-6] #### File: FACS.xlsx
 **Description:**  **Sheet 1: Appendix** **TABLE 1**- EFFECT OF 2 HOURS OF
 PCP ON SERUM PROTEINS ON DAY 1 AND ON DAY 30. Each entry is the percent
 increase in the serum concentration of a given protein after 2 hours of
 PCP on the specific day. Statistically significant elevation in MCP-1 and
 IL-6 were obseserved after two hours of PCP on Day 1 only. **TABLE 2**-
 EFFECT OF 30 DAYS OF PCP (THREE HOURS DAILY).The percent increase in the
 serum concentration of a given protein at 30 days (referenced to Day 1) is
 tabulated before (T=0) and after 2 hours of PCP (T=2hrs). Statistically
 significant elevation in TNF-alpha were observed on Day 30 compared to Day
 1 before starting the 2 hour PCP session. **TABLE 3**- EFFECT OF 2 HOURS
 OF PCP ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UC data). Column
 headings "DAY 1": Prior to Filgrastim, "5th dose":1
 day after the 5th dose of Filgrastim, "10th dose": 1 day after
 the 10th dose of Filgrastim. Each entry is the percent increase in the
 serum concentration of a given protein after 2 hours of PCP on the
 specific day. Two hours of observed PCP did not statistically influence
 the concentration of these proteins. **TABLE 4**- EFFECT OF FILGRASTIM ON
 SERUM PROTEINS IN THE FILGRASTIM+PCP group (UC data). Column headings:
 "5th dose": 1 day after the 5th dose of Filgrastim, "10th
 dose": 1 day after the 10th dose of Filgrastim. Each entry is the
 percent increase in the serum concentration of a given protein at start
 (T=0), and at end of 2 hours of PCP (T=2hrs) on each of those days.
 Percent in Columns B and C are relative to T=0 and T=2hrs on Day 1. The P
 values were derived from paired TTest comparing the ELISA concentrations
 before and after PCP use one day after the 5th FIlgrastim Dose
 ("B") and one day after the 10th Filgrastim dose ("C")
 to the Day 1 ELISA concentrations before and after PCP use.. Statistically
 significant elevation in Plasmin, FDP, HGF,MMP-9 were observed on both
 days. Statistically significant elevation in PDGF-AA, PDGF-BB, IGF-1,
 TGFbeta, and TNF-alpha were observed at one time point. Statistically
 significant drop in MCP-1 was observed at one time point prior to starting
 the 2 hour PCP session. **TABLE 5**- EFFECT OF 2 HOURS OF PCP ON SERUM
 PROTEINS IN THE FILGRASTIM+PCP group (UIC data) ON DAY 1, 14 AND 29. Each
 entry is the percent increase in the serum concentration of a given
 protein after 2 hours of PCP on the specific day. Two hours of observed
 PCP did not statistically influence the concentration of these proteins.
 Pvalues were derived from the the paired TTest comparing ELISA
 concentrations before and after PCP use on each Day. **TABLE 6**- EFFECT
 OF FILGRASTIM ON SERUM PROTEINS IN THE FILGRASTIM+PCP group (UIC data).
 Each entry is the percent increase in the serum concentration of a given
 protein at start (T=0), and at end of 2 hours of PCP (T=2hrs) on each of
 those days. Percent on Days 14 and 29 are relative toDay 1, at T=0 and
 T=2hrs. The P values were derived from paired TTest comparing the ELISA
 concentrations before and after PCP use on Day 14, and Day 29, to Day 1
 ELISA concentrations before and after PCP use. Statistically significant
 elevation in Plasmin, FDP, HGF,VEGF-A, and MMP-9 were observed on both
 days. **TABLE 7**- EFFECT OF PCP ON SERUM NITRITE. The first 2 columns are
 percent nitrite increases on Day 1 and Day 30 before and after 2 hours of
 PCP derived from the ELISA concentrations before and after 2 hours of PCP
 on each day. The Pvalues are derived from the T-test comparing the ELISA
 concentrations. The 3rd and 4th columns show the cumulative effect of 30
 days of PCP. Each entry is the percent increase in serum nitrite before
 and after 2 hours on PCP on Day 30 relative to before after 2 hours on PCP
 on Day 1. The Pvalues are derived from the T-test comparing the ELISA
 concentrations. **TABLE 8**- SERUM NITRITE IN PATIENTS TREATED WITH
 FILGRASTIM AND PCP. The first 3 columns show the effect of 2 hours of PCP
 1)prior to Filgrastim ("DAY 1"), 2) One day after the 5th dose
 of Filgrastim ("B"), and 3) One day after the 10th dose of
 Filgrastim ("C"). Each entry in the first 3 columns is the
 percent increase in serum nitrite after 2 hours of PCP. The P values are
 derived from a paired TTest of the serum nitrate values from blood drawn
 before and immediately after 2 hours of PCP. The last 4 columns show the
 effect of FIlgrastim relative to Day 1, both before and after 2 hours of
 PCP. The P values are derived from a paired TTest of the serum nitrate
 values from blood drawn on Day 1 and on Day 30 before and immediately
 after 2 hours of PCP. **TABLE 9**-CYTOMETRY IN PATIENTS TREATED WITH PCP
 ALONE **TABLE 10**-CYTOMETRY IN PATIENTS TREATED WITH FILGRASTIM AND PCP
 **TABLE 11**-CYTOMETRY IN PATIENTS TREATED WITH FILGRASTIM AND PCP.
 Percent increase in CD31, CD34, and VEGFR2 cell counts after 2 hours of
 PCP use on Day 1, and 24 hours after the 5th and 10th doses of Filgrastim.
 The pValues are derived from the paired TTests compairing cell counts at
 T=0 to those at T=2 hours). The results show small but significant
 increases in circulating CD31, CD34 and VEGFR2. **TABLE 12**-CYTOMETRY IN
 PATIENTS TREATED WITH FILGRASTIM AND PCP. The influence of Filgastim on
 the percent increase in CD31, CD34, and VEGFR2 cell counts 24 hours after
 the 5th (Column B) and 10th (Column C) doses of Filgrastimare tabulated
 (relative to Day 1 prior to Filgrastim dose). Entries were derived from
 the nitrite in blood drawn before (T=0) and then after 2 hours of PCP (T=
 2 hrs) on each day and compared to the same times on Day 1. The pValues
 are derived from paired TTests compairing cell counts at T=0 to those at
 T=2 hours on each day. Significant increases in circulating CD31, CD34 and
 VEGFR2 were identified. **Sheet 2: APPENDIX TABLES** Tabbles: PCP ALONE
 ELISA TABLES, FILGRASTIM + PCP ELISA TABLES, CYTOMETRY PCP ALONE,
 CYTOMETRY FILGRASTIM + PCP, NITRITE: PCP ALONE, NITRITE (nM) FILGRASTIM +
 PCP, and PCP alone for 30 days. **Sheet 3: FACS** Fluorescence Activated
 Cell Sorting plots for CD31, CD34, VEGFR2 for each patient at enrollment
 and then one day after the fifth and one day after the 10th Filgrastim
 dose. These data are from the university-affiliated institution in
 patients who received Filgrastim. ## Human subjects data The human data
 obtained from the University affiliated hospitals were obtained and
 published with each patient's consent.