An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.

Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions.

To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κß was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Vitronectin Levels in the Plasma of Neuroblastoma Patients and Culture Media of 3D Models: A Prognostic Circulating Biomarker?

Vitronectin is a glycoprotein present in plasma and the extracellular matrix that is implicated in cell migration. The high amount of vitronectin found in neuroblastoma biopsies has been associated with poor prognosis. Moreover, increased vitronectin levels have been described in the plasma of patients with different cancers. Our aim was to assess vitronectin as a potential circulating biomarker of neuroblastoma prognosis. Vitronectin concentration was quantified using ELISA in culture media of four neuroblastoma cell lines grown in a monolayer and in 3D models, and in the plasma of 114 neuroblastoma patients. Three of the neuroblastoma cell lines secreted vitronectin to culture media when cultured in a monolayer and 3D models. Vitronectin release was higher by neuroblastoma cells cultured in 3D models than in the monolayer and was still elevated when cells were grown in 3D scaffolds with cross-linked vitronectin. Vitronectin secretion occurred independently of cell numbers in cultures. Its concentration in the plasma of neuroblastoma patients ranged between 52.4 and 870 µg/mL (median, 218 µg/mL). A ROC curve was used to establish a cutoff of 361 µg/mL, above which patients over 18 months old had worse prognosis (p = 0.0018). Vitronectin could be considered a new plasma prognostic biomarker in neuroblastoma and warrants confirmation in collaborative studies. Drugs inhibiting vitronectin interactions with cells and/or the extracellular matrix could represent a significant improvement in survival for neuroblastoma patients.

Periosteal Bone Formation Varies with Age in Periostin Null Mice.

Periostin, also known as osteoblast-specific factor 2, is a matricellular protein predominantly expressed at the periosteum of bone. During growth and development, periostin contributes to periosteal expansion by facilitating osteoblast differentiation and mineralization. Later in life, periosteal expansion provides an adaptive strategy to increase tissue strength without requiring substantial increase in bone mass. However, the function of periostin past skeletal maturity and during advanced aging is relatively unknown. The objective of this study was to examine the function of periostin in maintaining bone mass and tissue strength across different ages. In periostin null mice (Postn-/-), periosteal bone formation was significantly reduced in young (3 months) and adult mice (9 months). The lack of bone formation resulted in reduced bone mass and ultimate strength. Conversely, periosteal bone formation increased at advanced ages in 18-month-old Postn-/- mice. The increase in periosteal mineralization at advanced ages coincides with increased expression of vitronectin and osteopontin. Periosteal progenitors from Postn-/- mice displayed an increased capacity to mineralize when cultured on vitronectin, but not type-1 collagen. Altogether, these findings demonstrate the unique role of periostin in regulating periosteal bone formation at different ages and the potential for vitronectin to compensate in the absence of periostin.

A vitronectin-derived dimeric peptide suppresses osteoclastogenesis by binding to c-Fms and inhibiting M-CSF signaling.

Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.

Calcium and hydroxyapatite binding site of human vitronectin provides insights to abnormal deposit formation.

The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.

Calcium-induced environmental adaptability of the blood protein vitronectin.

The adaptability of proteins to their work environments is fundamental for cellular life. Here, we describe how the hemopexin-like domain of the multifunctional blood glycoprotein vitronectin binds Ca2+ to adapt to excursions of temperature and shear stress. Using X-ray crystallography, molecular dynamics simulations, NMR, and differential scanning fluorimetry, we describe how Ca2+ and its flexible hydration shell enable the protein to perform conformational changes that relay beyond the calcium-binding site and alter the number of polar contacts to enhance conformational stability. By means of mutagenesis, we identify key residues that cooperate with Ca2+ to promote protein stability, and we show that calcium association confers protection against shear stress, a property that is advantageous for proteins that circulate in the vasculature, like vitronectin.

Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases.

Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood-brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer's disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood-brain barrier and neurodegenerative diseases.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Selective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces.

In orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents optimal integration of the prostheses with the surrounding bone. To overcome these issues, biomimetic titanium implants have been developed where synthetic peptides have been selectively grafted on titanium surfaces via Schiff base formation. We used the retro-inverted sequence (DHVPX) from [351-359] human Vitronectin and its dimer (D2HVP). Both protease-resistant peptides showed increased human osteoblast adhesion and proliferation, an augmented number of focal adhesions, and cellular spreading with respect to the control. D2HVP-grafted samples significantly enhance Secreted Phosphoprotein 1, Integrin Binding Sialoprotein, and Vitronectin gene expression vs. control. An estimation of peptide surface density was determined by Two-photon microscopy analysis on a silanized glass model surface labeled with a fluorescent analog.

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin.

Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvß3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvß3 integrin.

Correlations between vitronectin, miR-520, and miR-34 in patients with stenosis of coronary arteries.

BACKGROUND: In-stent restenosis usually occurs by platelet activation, neointima formation, VSMC migration, and proliferation in the position of the vessel stent. The monocytes have a magnificent role in neointimal hyperplasia since these cells recruit to the site of vessel injury through chemokines and other secretion proteins. This study is focused on the investigation of vitronectin, miR-193, miR-34, and miR-520 expression levels in PBMCs isolated from stenosed patients. METHODS: A total of sixty subjects undergoing coronary artery angiography containing patients with stent no restenosis (n = 20), in-stent restenosis (n = 20), and healthy participants (n = 20) participated in the study. The vitronectin, miR-193, miR-34, and miR-520 expression levels were measured by the RT-qPCR technique. Data were analyzed by SPSS software. RESULTS: The vitronectin, miR-34, and miR-520 expression levels changed significantly in patients with vessel in-stent restenosis (p = 0.02, p = 0.02, and p = 0.01, respectively). Furthermore, there were inverse correlations between the expression levels of vitronectin gene and miR-34 (r = - 0.44, p = 0.04) as well as miR-520 (r = - 0.5, p=0.01). CONCLUSIONS: The molecular events in the vessel stenosis may be affected by targeting vitronectin with miR-520 and miR-34.

Integrin αv and Vitronectin Prime Macrophage-Related Inflammation and Contribute the Development of Dry Eye Disease.

Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.

Blood Vitronectin Induces Detrimental Brain Interleukin-6 and Correlates With Outcomes After Stroke Only in Female Mice.

Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than hormones. We investigated the role of the blood protein VTN (vitronectin) after ischemic stroke in mice. Methods- Adult male and female VTN knockout and wild-type littermates and C57BL/6 mice received a middle cerebral artery occlusion and the injured brain tissue analyzed 24 hours to 3 weeks later for cell loss and inflammation, as well as neurological function. Blood VTN levels were measured before and after stroke. Results- Intravenously injected VTN leaked extensively from bloodstream into brain infarct and penumbra by 24 hours after stroke. Strikingly, VTN was detrimental in female, but not male, mice, as shown by reduced brain injury (26.2±2.6% versus 13.4±3.8%; P=0.018; n=6 and 5) and forelimb dysfunction in female VTN knockout mice. Stroke increased plasma VTN 2- to 8-fold at 24 hours in females (36±4 versus 145±24 µg/mL; P<0.0001; n=10 and 7), but not males (62±8 versus 68±6; P>0.99; n=10 and 7), and returned to control levels by 7 days. Individually variable VTN levels at 24 hours correlated with stroke-induced brain injury at 7 days only in females. VTN promoted stroke-induced microglia/macrophage activation and leukocyte infiltration in females. Proinflammatory IL (interleukin)-6 greatly increased in the striatum at 24 hours in wild-type mice but was increased ≈60% less in female (739±159 versus 268±111; P=0.02; n=7 and 6), but not male (889±178 versus 1179±295; P=0.73; n=10 and 11), knockout mice. In individual wild-type females, plasma VTN levels correlated with striatal IL-6 expression at 24 hours. The female-specific effect of VTN-induced IL-6 expression following stroke was not due to gonadal hormones, as shown by ovariectomy and castration. Lastly, intrastriatal injection of IL-6 in female mice immediately before stroke reversed the VTN knockout phenotypes of reduced brain injury and microglia/macrophage activation. Conclusions- VTN plays a novel sexually dimorphic detrimental pathophysiological role in females and might ultimately be a therapeutic target to improve stroke outcomes in women.

Mechanisms of integrin αVß5 clustering in flat clathrin lattices.

The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cell-extracellular matrix adhesion. The vitronectin-binding integrin αVß5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVß5 is predominantly found in FCLs, and formation of the αVß5-containing FCLs requires the presence of vitronectin as ligand, Ca2+, and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of ß5 and the cytoplasmic domains of ß1 or ß3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVß5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVß5 in FCLs is dictated by the ß5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the ß5 subunit cytoplasmic domains.

A Toll-like receptor 2-integrin beta3 complex senses bacterial lipopeptides via vitronectin.

Toll-like receptor 2 (TLR2) initiates inflammation in response to bacterial lipopeptide (BLP). However, the molecular mechanisms enabling the detection of BLP by TLR2 are unknown. Here we investigated the interaction of BLP with human serum proteins and identified vitronectin as a BLP-recognition molecule. Vitronectin and its receptor, integrin beta(3), were required for BLP-induced TLR2-mediated activation of human monocytes. Furthermore, monocytes from patients with Glanzmann thrombasthenia, which lack integrin beta(3), were completely unresponsive to BLP. In addition, integrin beta(3) formed a complex with TLR2 and this complex dissociated after BLP stimulation. Notably, vitronectin and integrin beta(3) coordinated responses to other TLR2 agonists such as lipoteichoic acid and zymosan. Our findings show that vitronectin and integrin beta(3) contribute to the initiation of TLR2 responses.

Complement C9 binding site and the anti-microbial activity of caprine vitronectin are localized in close proximity in the N-terminal region of the protein.

Vitronectin (Vn) is a ligand for complement C9 and modulates its activity that favors bacterial growth and survival. At the same time, the anti-microbial activity of the heparin-binding region of human Vn has been documented. To understand these diverse and opposite functions of the protein, we have analyzed the interaction of caprine Vn with C9 in the homologous system. In a previous study, the C9 binding activity was mapped to the N-fragment of the caprine Vn (N-Vn), representing the first 200 amino acids. Interestingly, this fragment also inhibited bacterial growth. In this study, we have generated four sub-fragments of N-Vn and analyzed C9 binding by ELISA, blot overlay, surface plasmon resonance and circular dichroism spectroscopy. These sub-fragments were also tested for antimicrobial activity against E. coli and S. aureus by drop plate method and analyzing cell death by flow cytometry. Results of these analyses together with previous data suggest that in addition to the second RGD motif (106-108 amino acids), the first 47 residues are also required for C9 binding. The anti-microbial tests employed indicate that the growth inhibitory property is contributed by 101-150 residues of Vn. These results provide an initial insight into two diverse Vn functions.

Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen.

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10-7-10-8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.

HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication.

Previous work has shown that the Tat protein of Human Immunodeficiency Virus (HIV)-1 is released by acutely infected cells in a biologically active form and enters dendritic cells upon the binding of its arginine-glycine-aspartic acid (RGD) domain to the α5ß1, αvß3, and αvß5 integrins. The up-regulation/activation of these integrins occurs in endothelial cells exposed to inflammatory cytokines that are increased in HIV-infected individuals, leading to endothelial cell dysfunction. Here, we show that inflammatory cytokine-activated endothelial cells selectively bind and rapidly take up nano-micromolar concentrations of Tat, as determined by flow cytometry. Protein oxidation and low temperatures reduce Tat entry, suggesting a conformation- and energy-dependent process. Consistently, Tat entry is competed out by RGD-Tat peptides or integrin natural ligands, and it is blocked by anti-α5ß1, -αvß3, and -αvß5 antibodies. Moreover, modelling-docking calculations identify a low-energy Tat-αvß3 integrin complex in which Tat makes contacts with both the αv and ß3 chains. It is noteworthy that internalized Tat induces HIV replication in inflammatory cytokine-treated, but not untreated, endothelial cells. Thus, endothelial cell dysfunction driven by inflammatory cytokines renders the vascular system a target of Tat, which makes endothelial cells permissive to HIV replication, adding a further layer of complexity to functionally cure and/or eradicate HIV infection.