People living with HIV (PLWH), even with anti-retroviral therapy (ART), have an accelerated and augmented onset of non-AIDS related diseases such as the premature development of cardiovascular disease (CVD). In fact, CVD has become the second most common cause of non-AIDS related mortality in PLWH in the US. Herein, we aim to decipher the underlying mechanism for HIV-associated CVD. Specifically, our focus is on the bone marrow-blood (BMB) barrier and stem cell niche, as a vulnerable microenvironment that regulates the immune status in CVD. The bone marrow (BM) is an important reservoir for hematopoietic stem cells (HSCs), which give rise to immune cells including circulatory monocytes (inflammatory vs non-inflammatory). Recent studies have highlighted the importance of vasculature permeability (or lack thereof) in controlling HSC differentiation. Additionally, areas of the vasculature in the BM niches responsible for maintaining the long-term HSCs exhibit restrictive permeability properties (regulated by pericytes) similar to that of the blood-brain barrier. While the effect of HIV infection in the BM in the era of ART is unknown, it may mirror what is observed in the brain. Of note, it is well established that HIV infection and inflammation in the brain leads to reduced pericyte coverage and increased vascular permeability. Epidemiological studies indicate that comorbid substance use disorder is common in PLWH. Furthermore, drugs of abuse are well documented in exacerbating HIV pathology. For example, chronic cocaine use independently increases CVD risk and further augments its development in PLWH, highlighting a synergistic link between HIV infection and cocaine use. We propose that increased BM vascular disruption and reduced pericyte coverage resulting from HIV infection and cocaine could alter the balance of HSC differentiation and drive the underlying chronic immune activation which advances CVD progression. Thus, our hypothesis is that HIV and cocaine induce BMB barrier dysfunction which skews HSCs towards differentiation and production of inflammatory monocytes that promote early CVD. The study of dysfunctional BM microenvironments has never been examined as a factor in CVD during HIV infection/drug use. Our approach is highly conceptually and technically innovative and would be the first to study changes in the BMB barrier. This hypothesis will be examined using tissue clearing, microCT and advanced imaging techniques to map the 3D vascular architecture in humanized HIV-infected mice. Finally, we propose to develop a new human 3D tissue engineered model of the BM vasculature for the study of HIV pathogenesis. In brief, chronic immune activation is considered the leading factor driving early CVD in HIV+/chronic cocaine users; however, the underlying cause remains unknown. Therefore, identifying the mechanism of immune activation could lead to targeted treatment of HIV+ patients/drug users and management strategies to slow or prevent plaq...