Vascular activation is characterized by increased proinflammatory, prothrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19, have been noted to have increased activation of the Janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. We sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells and leukocytes. By using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2^V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte–endothelial adhesion.

Chemicals and reagents

Ruxolitinib (CAS 941678-49-5, Cat#11609) and fedratinib (TG101348) were purchased from Cayman Chemical, dissolved in DMSO, and stored as 5 μl stock aliquots: 20 mM ruxolitinib at −20 °C and 10 mM fedratinib at −80 °C. Recombinant human TNF-α was obtained from R&D Systems (#210-TA). Nuclear extracts were prepared by using the kit from Cayman Chemical (#10009277) following the manufacturer’s guidelines. Protein was quantified by using the Pierce BCA Protein Assay Kit (ThermoScientific Cat#23225). Human factor X (#HCX-0050) and human factor VIIa (HCVIIA-0031) were purchased from Haematologic Technologies. Chromogenic substrate for the determination of factor Xa activity (Chromogenix S-2765, Diapharma) was reconstituted per manufacturer’s instructions and stored at 4 °C. The urokinase type plasminogen activator human chromogenic activity assay kit was purchased from Abcam (Cat#ab8915) and used following the manufacturer’s protocol. HEPES buffered saline [HBS] (20 mM HEPES, pH 7.4, 150 mM NaCl) was used for tissue factor assay with the addition of 3% w/v bovine serum albumin (Fraction V; Fischer). CaCl2 (10 mM, final concentration) was added. For quenching the reactions, EDTA (10 mM final concentration) was added.

Antibodies

The following antibodies were used for immunoblots: P-STAT1 Tyr701) (58D6) Rabbit mAb Cat#9167); STAT1 (D4Y6Z) Rabbit mAb #1499; P-Stat3 (Y705) (D3A7) Rabbit mAb #9145, STAT3 (79D7) Rabbit mAb#4904, P-cJun63 (Ser63)(54B3) Rabbit mAb #2361, and c-Jun (60A8) Rabbit mAb#9165; P-NF-kB p65 (Ser536) (93H1) Rabbit mAb #3033; NF-kB (D14E12) XP Rabbit mAb #8242; GAPDH (D4C6R) Mouse mAb #97166, P-p44/42 MAPK (T202Y204) (20G11) (P-Erk1/2) Rabbit mAb#4376; p44/42 MAPK (Erk1/2) Rabbit mAb #9102; P-Akt (Ser473) (D9E) XP Rabbit mAb #4060; Akt (pan)(C67E7) Rabbit mAb #4691; and P-p38MAPK(Thr180/Tyr182) (D3F9)XP Rabbit mAb #4511, all from Cell Signaling Technology.

The following antibodies were used for immunofluorsence*:* P-selectin (R&D Systems #AF737), VWF (vWF, Cedarlane #CL20176A-R), and VE-cadherin (R&D Systems #AF1002).

Endothelial cell culture

Primary human umbilical vein endothelial cells (HUVEC) were isolated as previously described [31]. For static cultures, cells were grown until 95% confluent and then changed to 0.1%-1.0% FBS media. All cells were between passages 2 and 5 for experiments.

Coagulation assays

For tissue factor (TF) generation, treated HUVECs were cultured for 30 minutes in the presence of anti-TF antibody (BD Biosciences) or isotype IgG control (BD Biosciences) along with recombinant factor VIIa (100 pM, final concentration), factor X (135 nM, final concentration Enzyme Research Laboratories), and 5 mM calcium in HBS-BSA buffer. After 30 minutes, the sample was quenched with EDTA, and chromogenic Xa generation was conducted by using 0.5 mM Chromagenix FXa and reading absorbance at 450 nm every 30 seconds on a plate reader (Molecular Technologies).

For uPA activity, HUVECs grown in 24-well plates were incubated in 1% FBS media with ruxolitinib (0.4 μM) or fedratinib (1 μM) JDB for 30 minutes followed by the addition of 10 ng/mL TNF-α (R&D Systems) JDB for 4 hours. Conditioned media were collected and spun at 1500 ×g at 4 °C for 15 minutes and stored at −80 °C. Conditioned media were assessed for uPA activity over 4 hours at 37 °C following the manufacturer’s direction (Abcam).

Microfluidics devices

Fabrication

A single-layer microfluidic device was fabricated as a platform to study leukocyte and platelet velocity. The device design included one long resistor with 8 straight sections, with one inlet port and one outlet port. The device consisted of a polymethyl siloxane (PDMS) microchannel layer (405 144 μm [Length] × 1000 μm [Width] × 50 μm [Height]) bonded to a glass substrate. The PDMS layer of the device was fabricated by using a soft photolithography mask designed using AutoCAD (Autodesk). A master mold was created by coating a negative photoresist onto a silicon substrate followed by UV light exposure and removal of excess material. The master mold was used to generate the PDMS-based channel layer. The PDMS layer and glass substrate were coated in O2 plasma for 2 minutes and then bonded together followed by the evaporation of excess water molecules through incubation for 1 hour on a hot plate set to 110 °C.

HUVEC culture in devices

Microfluidic devices were coated with 2% gelatin solution for 3 minutes at room temperature. ECs (passages 2-5) were trypsinized and resuspended in EGM-2 cell culture medium supplemented with a EGM-2 SingleQuots growth kit (Lonza) at a density of 1.0 × 10⁶ cells/μL. ECs were then pipetted into the inlet port of the device until all channels were evenly covered. Devices were incubated at 37 °C, 5% CO2 for 30 minutes to allow for cell adherence. In addition, 3-mL syringes (BD) were filled with EGM-2 and connected to 0.020 inch ID plastic tubing (Tygon). The plastic tubing was connected to the inlet port of the device, and the syringe was secured in a syringe pump (kd Scientific). The syringe pump was set to a flow rate of 50 μL/hour for 24 hours, and devices were place at 37 °C, 5% CO2. After 24 hours, 3-mL syringes were switched out for 60-mL syringes, and the flow rate was changed to 2 mL/h for the next 48 hours. After 48 hours, the devices were ready for subsequent treatment.

Treatment of devices

Before flowing blood through, endothelialized devices were treated by adding 10 ng/mL TNFα, 0.4 μM ruxolitinib, 1 μM fedratinib, JDB or combination treatment (Rux+TNF or Fed+TNF) into EGM-2 media followed by incubation for 4 hours at 37 °C, 5% CO2 with a flow rate of 2 mL/h (15 dyne/cm²). Each device corresponded to one treatment condition.

Fluorescence imaging

On the day of the experiment, blood samples were obtained and labeled with calcein live stain (BioLegend) to tag leukocytes and platelets, 15 minutes before running the sample. Labeling of cells was confirmed by using blood smear before loading the sample into the microfluidic device. The labeled sample was loaded into a 1 mL syringe (BD) and set into a syringe pump running at a flow rate of 150 μL/h (1 dynes/cm²). Endothelialized microfluidic devices were disconnected from the syringe pumps containing EGM-2 and reconnected to the 1 mL syringe containing patient blood on a microscope stage. As blood flowed through the devices, 10-second videos were captured of at least 2 nonoverlapping regions of interest (ROI)s along the 8 long, straight sections of the microchannels by using a fluorescent microscope (Zeiss) at 10× magnification. Camera speed was set to 6.4 frames per second, with an average of 64 frames per video. Video was recorded for 15 minutes per device, resulting in 12-20 videos captured per device.

Image analysis

Images were processed by using FIJI (National Institutes of Health). A FIJI plugin, TrackMate [32], was used to quantify the velocity of cells passing through the ROIs described above. This camera speed excludes single platelet behavior, and events were thresholded to include tracks with between 50 and 200 spots. The mean velocity of each “track” corresponding to one cell was recorded for all of the tracks in each video captured. For each condition, the average velocity was calculated from around 6 of the total number of videos captured. The SD was calculated for each treatment condition.