Testosterone Deficiency and Endothelial Dysfunction After Spinal Cord Injury

The vascular endothelium plays a central role in atherosclerotic cardiovascular disease and may contribute to the increased risk of myocardial infarction and stroke following spinal cord injury (SCI). Endothelial dysfunction is characterized by impaired vasodilator function and reduced fibrinolytic capacity.

Endothelium-dependent vasodilation is primarily mediated by nitric oxide (NO), which induces rapid relaxation of vascular smooth muscle. Fibrinolysis is the breakdown of thrombi within blood vessels, and is facilitated by endothelial cells through the synthesis and release of tissue-type plasminogen activator (t-PA). Importantly, endothelial dysfunction often precedes detectable atherosclerosis and predicts future major vascular events.

Low testosterone (T) is a common secondary complication that occurs early after SCI, with hypogonadism being four times more prevalent in men with SCI. Testosterone has known antioxidant properties and its deficiency may contribute to endothelial dysfunction. Testosterone deficiency may represent a modifiable risk factor for vascular impairment after SCI.

This cross-sectional study will include 48 adults with subacute (<6 months), motor-complete (AIS A/B) paraplegia (neurological level T3 or below). 24 with testosterone deficiency and 24 with normal T levels. Endothelium-dependent vasodilation and t-PA capacity will be assessed via intra-arterial infusion of vasoactive drugs, with total forearm blood flow measured using venous occlusion plethysmography.

• Intra-arterial Infusion of Vasoactive Agents (Diagnostic test): A catheter is placed in the brachial artery of the non-dominant arm, and small doses of vasoactive drugs acetylcholine, isoproterenol, sodium nitroprusside are infused. Forearm blood flow will be measured using venous occlusion plethysmography. The purpose of this procedure is to assess endothelium-dependent and independent vasodilation by stimulating different vascular pathways. The acetylcholine infusion is to test muscarinic receptor, nitro oxide dependent, endothelium-dependent vasodilation. Isoproterenol was selected to stimulate tissue plasminogen activator based on its specificity and effectiveness at eliciting local and rapid tissue plasminogen activator release in adult humans. Sodium nitroprusside infusion is to assess endothelium-independent vasodilation.
• Intra-arterial Vitamin C Infusion (Diagnostic test): Vitamin C, a potent antioxidant, will be infused into the forearm and forearm blood flow will be re-evaluated to determine whether oxidative stress contributes to endothelial dysfunction.
• Blood Sampling (Diagnostic test): Blood will be sampled from the antecubital vein (\~50 mL) for biomarker analysis. This is to assess circulating biochemical and molecular indicators of vascular health and inflammation including levels of endothelial cell derived microvesicles.

Primary outcomes

• Endothelium-dependent vasodilation [Measured at baseline (without acetylcholine) and immediately after each acetylcholine dose for 3-5 minutes.]: Total forearm blood flow with be measured by strain gauge venous plethysmography under baseline conditions and under pharmacological manipulation with acetylcholine at increasing concentrations (8, 16, 32ug/ml).
• Endothelium-independent vasodilation [Measured at baseline (without sodium nitroprusside) and immediately after each sodium nitroprusside dose for 3-5 minutes.]: Total forearm blood flow with be measured by strain gauge venous plethysmography under baseline conditions and under pharmacological manipulation with sodium nitroprusside at increasing concentrations (1, 2, 4ug/ml).
• Tissue plasminogen activator release [Measured at baseline and immediately after each isoproterenol and sodium nitroprusside dose for 3-5 minutes.]: Net endothelial release or uptake of t-PA and PAI-1 (both antigen and activity levels) at each dose of isoproterenol and sodium nitroprusside will be calculated as the product of the arteriovenous concentration gradient and the infused forearm plasma flow. Arteriovenous concentration gradients for both t-PA and PAI-1 antigen and activity for each subject (at each time point) will be determined by subtraction of the values measured in simultaneously collected venous and arterial blood samples
• Endothelial cell-derived microvesicles concentration [Baseline]: Endothelial cell-derived microvesicles will be collected from venous blood samples and counted used flow cytometry to determine a circulating concentration.
• Endothelial cell-derived microvesicles effects of human coronary artery endothelial cells nitric oxide bioavailability. [Baseline]: Endothelial cell-derived microvesicles will be sorted and collected by fluorescence-activated cell sorting (FACS) flow cytometry. The endothelial cell-derived microvesicles will be co-cultured with human coronary artery endothelial cells. Endothelial Nitric Oxide Synthase and phosphorylation sites of interest will be measured by intracellular protein expression quantification of whole cell lysates by capillary electrophoresis immunoassays. Nitric oxide production will be assessed by total nitric oxide and nitrate/nitrite parameter assays
• Endothelial cell-derived microvesicles effects of human coronary artery endothelial cells reactive oxygen species and antioxidant capacity [Baseline]: Endothelial cell-derived microvesicles will be sorted and collected by fluorescence-activated cell sorting (FACS) flow cytometry. The endothelial cell-derived microvesicles will be co-cultured with human coronary artery endothelial cells. Super oxide dismutase and catalase expression will be measured by intracellular protein expression quantification of whole cell lysates by capillary electrophoresis immunoassays. Intracellular oxidative stress will be assessed by ROS-Glo H2O2 assay.

Sex: Male, ages: 18 Years to 89 Years

Inclusion Criteria:

* Between ages 18-89 years of age
* Male Sex
* History of motor complete (AIS A/B) paraplegia (NLI T3 or Below)
* Time since injury <6 months at time of enrollment (Subacute injury)
* Testosterone Deficiency defined as < 300ng/dL

Exclusion Criteria:

* Overt cardiovascular disease assessed by a) medical history, b) physical examination c) electrocardiogram
* Anaphylaxis to betadine, lidocaine, iodine
* Active infection at time of enrollment.
* Recent surgery (<1 month) at time of enrollment.
* History smoking tobacco (currently or in the past 12 months)
* History of more than low-risk history of alcohol consumption
* History of drug abuse
* History of use of cardiovascular-acting (i.e. statins, beta-blockers) therapeutics
* History of other health habits, medications, and supplements that could influence the outcome measures deemed by principal investigators and investigative team.

• Andrew Park, MD, Principal investigator, Craig Hospital