Short Communication: Proangiogenic Hematopoietic Cells In Acute HIV Infection

Introduction

C irculating endothelial progenitor cells (EPCs) were described for the first time in 1997. ¹ After the publication of the initial report by Asahara et al., increasing interest has been focused on the understanding of their mobilization and function under several conditions; furthermore, the potential use of EPCs as a new strategy in regenerative medicine has been hypothesized. ¹ Several clinical studies have shown that EPCs levels and functions are impaired in some pathological conditions. ^2,3

Putative endothelial progenitors include different cell populations with specific functional properties and different lineage derivation. ⁴ The colony forming unit-endothelial cells (CFU-ECs), identified by adapting the original method described by Asahara et al., ^1,5 derive from the nonadherent fraction of circulating mononuclear cells, appear early in culture (less than a week), do not possess the ability to self renew, and do not directly contribute to the generation of new vessels. ⁶ In contrast to CFU-ECs, the endothelial colony-forming cells (ECFCs) grow from the adherent mononuclear cell fraction after 10 days of culture, possess self-renewal capacity, and, importantly, are able to form vascular structures in vivo. ⁶ In addition, unlike CFU-ECs, which coexpress hematopoietic and endothelial surface antigens, ECFCs have a pure endothelial phenotype, and therefore they constitute the “true” circulating EPCs. ⁶ Nonetheless, it is widely accepted that CFU-ECs also provide a fundamental contribution to vascular repair in vivo, by secreting paracrine growth factors, which recruit ECFCs at the ischemic sites and orchestrate their proliferation. ^6,7 We recently demonstrated that HIV infection induces a significant reduction of circulating CFU-ECs, while the number of ECFCs appears similar to that found in the general population. ⁸ Nevertheless, the trafficking of these progenitors during acute HIV infection is unknown. In this study we evaluated the levels of ECFCs and CFU-ECs in two subjects with acute HIV infection at diagnosis and during antiretroviral therapy (ART).

Materials and Methods

The two patients of the current report presented at the Department of Infectious Disease of the University “G. D'Annunzio” of Chieti, in Fiebig stage I/II, with negative HIV-1-specific antibodies (test ELISA and Western blot) and positive viral load. They reported unprotected heterosexual intercourse about 30–60 days before the diagnosis; they had no comorbidity. As suggested by some guidelines, ART was administered. ^9,10 Specifically, one patient received tenofovir-disoproxil-fumarate (TDF) with emtricitabine (FTC), efavirenz (EFV), and maraviroc (MRV) and the second one was treated with TDF+FTC+EFV and raltegravir (RAL). These therapies were continued for 6 months, until HIV-RNA became undetectable. This therapeutic strategy aimed at reducing the wide spread of the virus in the reservoirs and at obtaining a rapid reduction of viremia. Peripheral blood samples were collected at the time of diagnosis and again after 6 months of therapy. Thirty-two healthy blood donors were included as a control group. The local ethic committees approved the study, which was conducted according to the Helsinki declaration, and all the participating subjects signed a written informed consent before enrollment.

Results

Data obtained in the two patients and the corresponding normal ranges observed in the control group are reported in Table 1. The frequencies of CFU-EC at diagnosis in the two patients were 0.25 and 0.1/2.5×10⁵ plated cells, respectively. These values were markedly lower than those found in normal subjects (9.8±2.4/2.5×10⁵ plated cells, median number±SEM). After 6 months of ART, the CFU-EC frequency increased in both patients (2.2 and 6.2/2.5×10⁵ cells, respectively). The median ECFC frequency in the control group was 0.1±0.04/10⁷ plated cells. Thirteen out of the 32 control samples showed no ECFC growth, in agreement with previous reports from our and other laboratories. ⁸ One patient showed no ECFC growth at diagnosis and after therapy. The other patient presented a high ECFC frequency at diagnosis (1/10⁷ plated cells), while ECFCs became undetectable after 6 months of ART.

The number of proviral DNA copies integrated in cells collected from CFU-ECs was higher at the time of diagnosis than after ART in both patients, while the search for HIV proviral DNA was negative in ECFC, confirming our previous observations ⁸ (Table 1). We also observed a decrease in the mean contents of APOBEC-3A, -3G, and -3F in CFU-ECs isolated from the two patients, which were 2.1, 2.3, and 2.2 at diagnosis and 0.4, 0.4, and 0.2 after 6 months of ART (results expressed as the ratio between APOBEC RNA and β-actin RNA, data not shown). Along with these findings we also observed the rise of CD4⁺ cells and the reduction of HIV-RNA, which became undetectable in both patients (Table 1). After 6 months of therapy, metabolic parameters worsened in both patients, with a relevant increase of TCh and of TGs, although we observed an improvement in hsCRP levels (Table 1). In addition, we found an increase in the HOMA-IR patient, whereas HDL cholesterol and cystatin C serum levels did not show significant modifications (Table 1). Finally, we did not observe any modification of hepatic and renal function indexes.

Discussion

To our knowledge, the behavior of various subsets of endothelial progenitors during acute HIV infection has never been evaluated to date. This study conducted in two patients showed that CFU-ECs, i.e., the hematopoietic proangiogenic progenitors, are infected by HIV at disease onset, resulting in the early and severe depletion of these progenitors since the initial phase of infection. Studies evaluating the impairment of EPCs during HIV infection so far reported debatable results, ^8,12 although the conflicting findings may result from the use of different methodologies for evaluating these cells. Specifically, studies quantifying EPCs by phenotype analysis report no differences between the general population and HIV-infected persons. ^12,13 In contrast, those studies employing the functional cell culture approach evidenced a significant perturbation of different EPC subsets in HIV-positive patients. ^14,15 Our findings in patients with acute HIV infection confirm that HIV infects and depletes the CFU-EC population while it does not affect the ECFC compartment, and further suggest that an impairment of endothelial repair function may be present even in the very early stages of the infection. Interestingly, a low level of CFU-ECs is a strong predictor of cardiovascular disease in the general population. ⁵

The optimal use of ART in patients with acute infection is unclear. ⁹ A few small studies showed some benefits when therapy is provided before or during seroconversion, with some degree of immune preservation and with sustained reduction of blood viral load after ART discontinuation. ^10,16 In the most recent treatment guideline it is argued that potential benefits to public and individual health may even justify the treatment of patients with acute HIV infection. ¹⁷ In our patients ART was started at diagnosis of acute infection: this approach led to a significant reduction of plasma viremia as well as to an increase of CD4⁺ cells and of CFU-EC levels. However, it is unclear whether these modifications will persist after ART discontinuation. An interesting finding emerging from this study is the increase of CFU-EC levels during ART despite the worsening of the lipid profile. This observation is apparently in contrast to the reduction of CFU-ECs reported in the general population with high cardiovascular risk, ⁵ although it strongly suggests that in the setting of HIV infection EPC trafficking is also markedly influenced by inflammatory and immunologic factors.

The mechanism by which APOBEC, particularly APOBEC-3G, inhibits HIV replication is not fully understood. The importance of APOBEC in the pathogenesis of HIV-related disease is highlighted by the fact that genetic variation in the locus encoding APOBEC3G can predict disease progression in HIV-infected people. ¹⁸ We recently found that both normal and HIV-infected CFU-EC contain very low amounts of ABOPEC. ⁸ Here we show that after 6 months of therapy the APOBEC levels in CFU-ECs are even lower than what found at the time of diagnosis. Nonetheless, it could be hypothesized that at infection onset the high viral load and the presence of high amounts of Vif stimulate APOBEC production, while thereafter, with the decline of HIV RNA copies, APOBEC production decreases. Finally, we observed that ART does not induce the disappearance of HIV in the restored circulating CFU-EC population, suggesting that this cell pool persists as a circulating reservoir of the virus.

In conclusion, this study demonstrates that HIV impairs the CFU-EC compartment very early and that ART is able to restore it. However, HIV-positive CFU-ECs persist in the circulation regardless of the decay of plasma viremia. Considering the high prevalence of cardiovascular diseases in HIV-infected people, ^19,20 this study encourages further studies on larger series of patients to specifically assess the involvement of proangiogenic cell populations in the pathogenesis and prevention of HIV-related cardiovascular diseases.