In our study we decided to use adult stem cells such as hematopoietic progenitor cells (CD34+) as our primary outcome measure . Our laboratory group has used hematopoietic cells as a cell based diagnostic and prognostic biomarker in multiple scenarios and have evaluated ECD evaluation modalities such as arterial stiffness measures , flow mediated dilatation and podocyte inflammatory markers [14-17].
This is a cross-sectional observational study examining patients with HIV and a history of taking either INSTI or NNRTI regimens for at least one year. The study was conducted in accordance with Good Clinical Practice guidelines set forth by the International Conference of Harmonization as well as local regulatory guidelines with the approval and oversight of the George Washington University Institutional Review Board.
Subjects were initially pre-screened to assess eligibility. Once determined preliminary eligibility, they were brought in for a screening visit to confirm eligibility via interview, medical record check and laboratory workup once the subject signed the informed consent form.
The study entailed a single timepoint visit. The assessments that were completed include: vital measurements such as BMI, blood pressure, heart rate, adverse event (AE) checks, and a peripheral blood draw. Approximately 60 ml of blood was drawn for CD34+ endothelial progenitor cell harvesting and routine blood work. Other parameters tested were measurement of waist-to-hip ratio, Tanita body composition scale, pulse wave analysis and pulse wave velocity to determine arterial stiffness.
A follow-up phone call visit was completed 30 days from the last in-person visit to assess for any residual adverse events (AE).
The subjects were included if they were males between 40 and 70 years old. Patients with BMI between 25 and 39.9 kg/m2 were included. We avoided subjects with severe obesity (BMI ≥ 40) as those subjects are deemed to have pre-existing CVD risk). Patients with normal and mildly impaired renal function were included, with lowest eGFR cut-off of 50 ml/min/1.73 (GFR, as calculated by MDRD formula).
Any patients with uncontrolled hyperglycemia, history of liver disease, clinically significant RBC cell disorders, HBV or HCV, chronic malabsorption, statin medication use, use of consistent steroid medications, untreated thyroid disease was excluded. Additional Inclusion and Exclusion criteria can be found in the Appendix.
The objective is to detect differences between INSTI and NNRTI based HAART based regimens Our primary outcome measures were cellular outcome measures pertaining to the cardiovascular-hematopoietic system with secondary outcome measures being measures of vascular function such as Arterial Stiffness [measured by pulse wave analysis (Augmentation Index) and pulse wave velocity (m/s)], and Serum Biochemistry Analysis pertaining to cardio-metabolic function parameters: such as Hemoglobin A1C(HbA1C) (as a reflection of glycemic control), Fasting Lipid Profile, C-Reactive Protein (inflammation), Adiponectin (measure of endothelial paracrine function), Fasting Insulin levels (measure of insulin resistance), and Interleukin-6 (inflammation).
The cell-based measures evaluating the function of CD34+ cells were to ascertain the effects of NNRTI and INSTI based regimens on CD34+ cell number, (CD34+ cell number percentage, (%CD34+ve out of total mononuclear cells, MNC, population), CD34 + cell function (cell migration function in response to a chemotactic agent, stromal derived factor-1a, SDF1α) and CD34+ cell gene expression (specifically genes related to endothelial function) in patients with HIV.
Secondary Outcome Measures
Body composition measurement
Body composition was measured manually as well as using Tanita™ BF-350 Body Composition Scale (an impedance scale). Manual measurement included height, waist circumference, hip circumference and BMI calculation. Tanita scale (Tanita Corporation of America, Inc, USA) uses a bio-impedance electrical impulse to measure body fat percent, fat mass (kg), fat-free mass (kg), percent body water, and water mass (kg) alongside weight. It also calculates the subject’s BMI and estimated basal metabolic rate (BMR).
This parameter was measured using Atcor Sphygmocor CP system (Atcor Technologies, USA). We obtained two outcomes: pulse wave velocity and pulse wave analysis.
Pulse wave analysis (PWA) was measured on the left Radial Artery with the subject supine. At least three readings were taken with Operator Index ≥ 80. Measurement includes Augmentation Index (AI), Augmentation Index adjusted for Heart Rate of 75 (AI-75), Augmentation Pressure (AP), Aortic and Radial reading of systolic, diastolic, pulse pressure and mean pressure.
Pulse wave velocity (PWV) was measured with the subject supine. This measurement requires a distal and proximal artery point delineation. Right femoral artery was used as the distal point with proximal being the left carotid. A straight line if drawn between these two points would include the heart. Index and ring fingers were used to manually localize the pulse, sometimes an arterial Doppler was used to localize the femoral pulse on patient with challenging body habitus. Once a stable pulse waveform was observed, the probe position was kept stable for 20 more pulses before the reading was finalized. Three readings were taken with standard deviation of less than 10%. The result reported a velocity in m/s, with standard error of the mean.
Biological sample and vital statistics collection
A venous blood sample was collected from the Antecubital fossa. About 60mL of blood was collected, centrifuged at 4°C, and plasma stored at -80°C until analysis or sent immediately to LabCorp. 60 ml for EPC analysis and 20 ml for standard of care blood tests which included Basic Metabolic Panel, Lipid Panel, HbA1c, fasting glucose, hsCRP, IL6, Adiponectin and Insulin. Urine sample was collected for urine Microalbumin and Creatinine ratio. Vital statistics were measured on the left arm including systolic pressure, diastolic pressure and heart rate, and long sublingual temperature.
Cellular and Clinical Assessments
CD34+ Endothelial Progenitor Cell Analysis
Peripheral blood samples (approximately 60 ml) were drawn from patients and phosphate buffered saline (1:1) was added. Identification and quantification of circulating cell phenotypes was performed on fresh blood samples, within 3 hours after collection, using flow cytometry. Briefly, mononuclear cells (MNCs) were then isolated from whole blood using a Ficoll density centrifuge method. MNCs were counted and an aliquot was used for CFU-Hill colony formation assay following the manufacturer’s instruction (Stem Cell Technologies, Vancouver, BC, Canada). Colony forming units were counted at day14. A fraction of the MNC were stained with fluorescein isothiocyanate (FITC)-conjugated antihuman CD34, Allophycocyanin (APC) conjugated antihuman CD184 (CXCR4), Allophycocyanin (APC) conjugated antihuman CD133 and FITC conjugated antihuman CD31 antibodies (Miltenyi Biotec GmbH, Bergisch-Gladback, Germany) in order to analyze specific progenitor cell surface markers (CD34 and CD133) and mature endothelial cell surface markers (CD31) or receptors for SDF1a ligand, CXCR4) by flow cytometry. After gating mononuclear cells in the side scatter (SSC)-A vs forward scatter (FSC)-A plot, CD34/CD33/CD184 single- and double-positive cells were identified. Cells were acquired on a fluorescence-activated cell sorter (FACS) Canto instrument (Becton Dickinson, USA) and scored with the Flo-Jo software.
To isolate EPCs (CD34+), MNCs were magnetically sorted through a column after cells were stained with CD34+ve microbeads antibody (Miltenyi Biotec GmbH, Bergisch Gladback, Germany). An aliquot of CD34+ cells were then stained with trypan blue and counted using an Auto Cellometer Mini (Nexcelom Bioscience, USA) to assess viability.
CD34+ gene expression analysis was performed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) as previously described (33). CD34+ cell total mRNA was extracted and purified using the RNeasy Minikit (Qiagen, Germany). mRNA was then converted into cDNA by using the high-capacity cDNA reverse transcriptase kit (Thermo Fisher Scientific, MA). Possible gene expression changes were assessed by a CFX96 real-time PCR systems (Bio-Rad, CA.) using Taqman Universal masters Mix II (Thermo Fisher Scientific, USA) and inventoried probes. The gene expression analysis included antioxidants, apoptosis, endothelial functions, chemotaxis, inflammation, and endothelial lineage cell surface markers. The expression of each individual gene was normalized to either housekeeping 18S and mean Cq values are reported.
The migratory capacity of CD34+ was evaluated using the CytoSelect 24-well Cell Migration Assay kit (Cell Biolads, Inc., San Diego, CA). Cells were suspended in Serum free media and seeded at 100,000 cells per insert. Migration of the cells through a 3um polycarbonate membrane to the wells containing a serum-free media (control) and chemoattractant SDF-1α (10 or 100 ng/mL) (from Sigma-Aldrich, USA) was assessed after cells were kept overnight in incubator. Migratory cells were dissociated from the membrane and subsequently lysed and quantified by fluorescence (480 nm/530 nm) using CyQuant GR dye (Cells Biolabs, Inc, USA). The fluorescence ratios between cells exposed to the chemotactic factor and cells exposed to chemoattractant-free media (control) along the visits were used to analyze the migratory capacity of the cells.
Because of the small sample size in this pilot program study, the analysis emphasis is on descriptive statistics. Differences between the INSTI and NNRTI groups were compared by Mann-Whitney tests. Significance test results indicate rough markers of parameters which may be affected by treatment regimen and not as confirmation of treatment effects. To balance Type 1 and 2 error rates for this preliminary study, no adjustments were made for multiple testing.