Title : Strategy to promote neovascularization and fibrinolysis in vascular disease
Abstract:
Neovascularization (NV) via growth of collateral arteries (arteriogenesis) and capillaries, arterioles, and venules (angiogenesis) becomes impaired as ischemia from vascular disease progresses. Our premise is that overcoming hemodynamic and cellular obstacles to NV should yield an effective, durable, low?risk, home?based, inexpensive option to surgical or catheter revascularization. NV and segmental recanalization of chronically occluded arteries were observed in patients with chronic limb?threatening ischemia (CLTI) treated with Filgrastim, a granulocyte colony stimulating factor, every 72 h for up to a month, and an infra?geniculate programmed compression pump (PCP) for 3 h daily. Molecular evidence for fibrinolysis and NV was sought. CLTI patients were treated with PCP alone (N = 19), or with Filgrastim and PCP (N = 8 and N = 6, at two institutions). Enzyme?Linked Immunosorbent Assay was used to measure the plasma concentration of plasmin and of fibrin degradation products (FDP), and the serum concentration of proteins associated with NV. 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 1 day after the 5th and the 10th Filgrastim doses. Each blood draw occurred before and after 2 h of supervised PCP. Significant (p < 0.01) PCP independent increases in the plasma concentration of plasmin (>10?fold) and FDP (>5?fold) were observed 1 day after both the 5th and the 10th Filgrastim doses, compared to Day 1. Significant (p < 0.05) increases in the concentration of pro?angiogenic proteins (e.g., HGF, MMP?9, VEGF A) were also observed. Filgrastim at this novel dosimetry induced fibrinolysis without causing acute haemorrhage, 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.
Audience Take Away:
- Improving the biochemical and cellular environment in ischemic tissue is achievable.Doing so can:
- Restore the innate capability to improve blood flow
- Promote clot lysis in chronically occluded vascular beds
- Reduce the complexity required to promote neovascularization (eliminates the need to micromanage the biology)
- Reduce the need for invasive vascular procedures with variable durability
- Nature micro-manages the circulation until vascular disease becomes too advanced. Strategies to optimize the bio-synthetic environment in the ischemic tissue should restore this capacity. Trials using specific drugs, specific cell types, or specific devices have not yielded a clinically accepted revascularization strategy. This is the 1st report of a novel method that overcomes specific obstacles so that Nature’s synthetic processes may operate more efficiently. While the clinical model used is limb threatening ischemia, the results have implications for central vascular disease (heart, lung, brain, etc). This study opens the door for a new research initiative to optimize the environment so natural compensatory mechanisms can be harnessed to treat vascular disease.
- List all other benefits:
- The goal of our approach is to improve blood flow. Achieving limb salvage also requires assiduous wound care, infection control, nutrition, avoidance of trauma, smoking cessation, and patient compliance. Ischemic rest pain, coldness, and numbness typically begin to improve in the second week. Filgrastim period was 30 days. PCP was continued until rest pain resolved and wounds healed. The larger forefoot ischemic wounds required over 12 months to heal. There is a race between the rate NV and fibrinolysis occur and the rate of progression of the destructive effect of severe tissue ischemia. Whether filgrastim or a long-acting form of filgrastim (e.g. peg-filgrastim) should continue for more than a month will need further investigation. As with all vascular interventions, the earlier the intervention the better. Improved blood flow was associated with limb salvage. The longest “no?option” patient that achieved limb salvage was treated in 2008 and remained amputation free at last follow-up in 2021. The proteomic, biochemical and cytometry data support the observed clinical benefit, angiographic observations, and confirmatory hemodynamic testing data. The study provides a foundation for refinement of the approach. As more experience is gained, and efficacy and durability is confirmed, this approach may become the first line treatment for patients with severe ischemia, even if amenable to invasive revascularization treatments. Below are opportunities for research and education.
- Fibrinolysis: An association between filgrastim and fibrinolysis was previously reported but was never evaluated clinically. [i] ,[ii],[iii]., Filgrastim, has a half-life of 3.5 hours. Our measurements were a day after the Filgrastim dose. Whether the increased plasmin is due to the increase in neutrophils, or to a specific change in the thrombostatic milieu will need further investigation. Furthermore, confirmation of the plasmin effect will require measuring the plasma concentrations of plasmin?alpha 2 antiplasmin [iv] and of tissue plasminogen activator? plasmin activator inhibitor complexes (tPA?PAI1) [v] in patients with chronic limb threatening ischemia (CLTI). Also, any contribution to the protein concentrations from the local tissue response to subcutaneous injection will require future placebo comparison.
- Our clinical discovery offered an unexpected solution to the management of chronic thrombus. Spontaneous recanalization of chronic thrombus is rare in CLTI. Moreover, it is unlikely to be achieved with prolonged intravascular infusion of potent fibrinolytic agents without the risk of hemorrhage. An agent that can safely lyse chronic obstructive thrombus over a period of weeks at a physiologic level is a potentially transformative therapy. Such a novel strategy would facilitate management of chronic ischemia not only in CLTI, but also in other vascular tissue beds (heart, brain, lung, kidney, etc.). While PCP is not feasible in central ischemic beds, it may not be needed due to proximity to the force of the pumping heart. Whether other forms of mechanical force can help provide a hemodynamic stimulus (e.g. ultrasound) to activate the endothelium to induce arteriogenesis would need further investigation.
- Filgrastim and VEGF 165b: The VEGF 165b isoform inhibits VEGFR2 signaling by inducing differential phosphorylation and has been reported to block angiogenesis in vivo [vi]. A monoclonal isoform-specific antibody against VEGF 165b was recently reported to promote nitric oxide independent therapeutic angiogenesis in a preclinical ischemia model [vii] . To assess the effect of Filgrastim on VEGF-165b expression in CLTI, its ELISA serum concentration was measured on Day 0, and then one day after the 5th and 10th doses in 8 CLTI patients. It was not detected in the 8 patients at baseline, nor after the 5th dose. In only one patient was it detected after the 10th dose (52+13 pg/ml). He had presented with diffuse tibial artery occlusive disease and toe gangrene. Deterioration in ischemic symptoms was observed at the end of the fourth week of the Filgrastim regimen, despite early improvement. A 20% drop in hemoglobin was detected at the time of the 10th dose, compared to before the first filgrastim dose. Occult blood in the stool (melena) led to the diagnosis of stage 4 adenocarcinoma of the stomach. The ELISA concentration of plasmin increased 4300% and 6733% after the 5th and 10th doses compared to baseline. Concurrently the ELISA FDP concentration increased 722% and 678% from baseline. The required hematology/oncology screens for hypercoaguability and for neoplasm prior to management of CLTI with Filgrastim were insufficient to make the oncologic diagnosis (exclusion criterion). The tumor did not respond to Folinic acid, Fluorouracil, and Oxaliplatin. It did respond to Taxol and Cyramza (anti-angiogenic) and was undetectable for nearly a year, though during this regimen ischemia progressed in both legs, culminating in unilateral below knee amputation. After chemotherapy the circulation in the remaining leg improved. However the tumor recurred briskly one year later and was unresponsive to further treatment. Filgrastim has been used in the oncologic population for decades, and has not been associated with promoting tumor growth, despite the pro-neovascularization and pro-fibrinolysis properties we observed. Whether Filgrastim induction of VEGF-165b tumor expression is the reason for this requires further investigation. The fibrinolytic effect may have contributed to the melena, which directly led to detection of this virulent tumor.
- Filgrastim resistance: Stem cell mobilization with G?CSF may not be effective in 15%–20% of patients, particularly in diabetics [viii]. Diabetics are also prone to accelerated atherosclerosis leading to CLTI. The concentration of proteins associated with fibrinolysis and NV in patients with this G?CSF resistance is yet to be delineated. Our patient cohort was too small to accurately observe differential influence of diabetes [ix]. Plerixafor (Mozobil, Genzyme) binds to CXCR4 and blocks the binding of its cognate ligand CXCL12 (Stroma?derived factor 1 alpha) [x] . The combination of G? CSF with plerixafor showed promise in overcoming ineffective hematopoietic stem cell mobilization and may be a solution to this problem if it arises in CLTI.
- There are no data yet on synergy of our approach with other agents (e.g. Growth hormone, anabolic agents), or devices (e.g. epidural), or modalities (ultrasound) or therapies (e.g. hyperbaric oxygen).
- Role after resolution of Vasculitis: An interesting observation is that two patients with CLTI following vasculitis (outside our reported patient cohort) had dramatic resolution of their ischemic symptoms (amputation free survival at 9 years) despite tissue loss and progression prior to treatment. One had Buerger’s vasculitis, the other marijuana associated vasculitis. Both were treated after smoking cessation and vasculitis resolution. Whether our findings related to NV and fibrinolysis by Filgrastim can be used to manage pulmonary hypertension following cessation of the vasculitis from COVID (long COVID) needs to be ascertained.
- Immunologic deficit and Wound healing: Filgrastim amplifies cellular immunity after cytotoxic chemotherapy. Diabetes impairs the immune system. In a 2013 Cochrane review of diabetic foot infections (not CLTI), Filgrastim was reported to reduce the need for surgical interventions, especially amputations, as well as the duration of hospitalization [xi]. The role of Filgrastim in assisting in the management of infection in CLTI wounds will need further investigation at the novel dosimetry we used.
- Next steps: With these favorable preliminary data, a controlled clinical trial is indicated. Previous trials of Filgrastim in the leg and in other circulatory beds (e.g., coronary) will need to be revisited at a dosimetry that capitalizes on both fibrinolysis and NV. In the lower extremity, increasing endothelial shear stress (for example with a PCP) to activate the endothelium and initiate arteriogenesis is important to overcome the hemodynamic obstacles to NV.
